Surgical irrigation solution and method for inhibition of pain and inflammation

ABSTRACT

A method and solution for perioperatively inhibiting a variety of pain and inflammation processes at wounds from general surgical procedures including oral/dental procedures. The solution preferably includes multiple pain and inflammation inhibitory at dilute concentration in a physiologic carrier, such as saline or lactated Ringer&#39;s solution. The solution is applied by continuous irrigation of a wound during a surgical procedure for preemptive inhibition of pain and while avoiding undesirable side effects associated with oral, intramuscular, subcutaneous or intravenous application of larger doses of the agents. One preferred solution to inhibit pain and inflammation includes a serotonin 2  antagonist, a serotonin 3  antagonist, a histamine antagonist, a serotonin agonist, a cyclooxygenase inhibitor, a neurokinin 1  antagonist, a neurokinin 2  antagonist, a purinoceptor antagonist, an ATP-sensitive potassium channel opener, a calcium channel antagonist, a bradykinin 1  antagonist, a bradykinin 2  antagonist and a μ-opioid agonist.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of copendingInternational Patent Application PCT/US95/16,028, filed Dec. 12, 1995,which designates the United States and which is a continuation-in-partof copending U.S. patent application Ser. No. 08/353,775, filed Dec. 12,1994, priority of the filing date of each application which are herebyclaimed under 35 U.S.C. § 120.

I. FIELD OF THE INVENTION

[0002] The present invention relates to surgical irrigation solutionsand methods, and particularly for anti-inflammatory, anti-pain,anti-spasm and anti-restenosis surgical irrigation solutions.

II. BACKGROUND OF THE INVENTION

[0003] Arthroscopy is a surgical procedure in which a camera, attachedto a remote light source and video monitor, is inserted into an anatomicjoint (e.g., knee, shoulder, etc.) through a small portal incision inthe overlying skin and joint capsule. Through similar portal incisions,surgical instruments may be placed in the joint, their use guided byarthroscopic visualization. As arthroscopists' skills have improved, anincreasing number of operative procedures, once performed by “open”surgical technique, now can be accomplished arthroscopically. Suchprocedures include, for example, partial meniscectomies and ligamentreconstructions in the knee, shoulder acromioplasties and rotator cuffdebridements and elbow synovectomies. As a result of widening surgicalindications and the development of small diameter arthroscopes, wristand ankle arthroscopies also have become routine.

[0004] Throughout each arthroscopy, physiologic irrigation fluid (e.g.,normal saline or lactated Ringer's) is flushed continuously through thejoint, distending the joint capsule and removing operative debris,thereby providing clearer intra-articular visualization. U.S. Pat. No.4,504,493 to Marshall discloses an isomolar solution of glycerol inwater for a non-conductive and optically clear irrigation solution forarthroscopy.

[0005] Irrigation is also used in other procedures, such ascardiovascular and general vascular diagnostic and therapeuticprocedures, urologic procedures and the treatment of burns and anyoperative wounds. In each case, a physiologic fluid is used to irrigatea wound or body cavity or passage. Conventional physiologic irrigationfluids do not provide analgesic, anti-inflammatory, anti-spasm and anti-restenotic effects.

[0006] Alleviating pain and suffering in postoperative patients is anarea of special focus in clinical medicine, especially with the growingnumber of out-patient operations performed each year. The most widelyused agents, cyclooxygenase inhibitors (e.g., ibuprofen) and opioids(e.g., morphine, fentanyl), have significant side effects includinggastrointestinal irritation/bleeding and respiratory depression. Thehigh incidence of nausea and vomiting related to opioids is especiallyproblematic in the postoperative period. Therapeutic agents aimed attreating postoperative pain while avoiding detrimental side effects arenot easily developed because the molecular targets for these agents aredistributed widely throughout the body and mediate diverse physiologicalactions. Despite the significant clinical need to inhibit pain andinflammation, as well as vasospasm, smooth muscle spasm and restenosis,methods for the delivery of inhibitors of pain, inflammation, spasm andrestenosis at effective dosages while minimizing adverse systemic sideeffects have not been developed. As an example, conventional (i.e.,intravenous, oral, subcutaneous or intramuscular) methods ofadministration of opiates in therapeutic doses frequently is associatedwith significant adverse side effects, including severe respiratorydepression, changes in mood, mental clouding, profound nausea andvomiting.

[0007] Prior studies have demonstrated the ability of endogenous agents,such as serotonin (5-hydroxytryptamine, sometimes referred to herein as“5-HT”), bradykinin and histamine, to produce pain and inflammation.Sicuteri, F., et. al., Serotonin-Bradykinin Potentiation in the PainReceptors in Man, Life Sci. 4, pp. 309-316 (1965); Rosenthal, S. R.,Histamine as the Chemical Mediator for Cutaneous Pain, J. Invest.Dermat. 69, pp. 98-105 (1977); Richardson, B. P., et. al.,Identification of Serotonin M-Receptor Subtypes and their SpecificBlockade by a New Class of Drugs, Nature 316, pp. 126-131 (1985);Whalley, E. T., et. al., The Effect of Kinin Agonists and Antagonists,Naunyn-Schmiedeb Arch. Pharmacol. 36, pp. 652-57 (1987); Lang, E., et.al., Chemo-Sensitivity of Fine Afferents from Rat Skin In Vitro, J.Neurophysiol. 63, pp. 887-901 (1990).

[0008] For example, 5-HT applied to a human blister base (denuded skin)has been demonstrated to cause pain that can be inhibited by 5-HT₃receptor antagonists. Richardson et al., (1985). Similarly,peripherally-applied bradykinin produces pain which can be blocked bybradykinin receptor antagonists. Sicuteri et al., 1965; Whalley et al.,1987; Dray, A., et. al., Bradykinin and Inflammatory Pain, TrendsNeurosci. 16, pp. 99-104 (1993). Peripherally-applied histamine producesvasodilation, itching and pain which can be inhibited by histaminereceptor antagonists. Rosenthal, 1977; Douglas, W. W., “Histamine and5-Hydroxytryptamine (Serotonin) and their Antagonists”, in Goodman, L.S., et. al., ed., The Pharmacological Basis of Therapeutics, MacMillanPublishing Company, New York, pp. 605-638 (1985); Rumore, M. M., et.al., Analgesic Effects of Antihistaminics, Life Sci 36, pp. 403-416(1985). Combinations of these three agonists (5-HT, bradykinin andhistamine) applied together have been demonstrated to display asynergistic pain-causing effect, producing a long-lasting and intensepain signal. Sicuteri et al., 1965; Richardson et al., 1985; Kessler,W., et. al., Excitation of Cutaneous Afferent Nerve Endings In Vitro bya Combination of Inflammatory Mediators and Conditioning Effect ofSubstance P, Exp. Brain Res. 91, pp. 467-476 (1992).

[0009] In the body, 5-HT is located in platelets and in central neurons,histamine is found in mast cells, and bradykinin is produced from alarger precursor molecule during tissue trauma, pH changes andtemperature changes. Because 5-HT can be released in large amounts fromplatelets at sites of tissue injury, producing plasma levels 20-foldgreater than resting levels (Ashton, J. H., et. al., Serotonin as aMediator of Cyclic Flow Variations in Stenosed Canine Coronary Arteries,Circulation 73, pp. 572-578 (1986)), it is possible that endogenous 5-HTplays a role in producing postoperative pain, hyperalgesia andinflammation. In fact, activated platelets have been shown to exciteperipheral nociceptors in vitro. Ringkamp, M., et. al., Activated HumanPlatelets in -Plasma Excite Nociceptors in Rat Skin, In Vitro, Neurosci.Lett. 170, pp. 103-106 (1994). Similarly, histamine and bradykinin alsoare released into tissues during trauma. Kimura, E., et. al., Changes inBradykinin Level in Coronary Sinus Blood After the ExperimentalOcclusion of a Coronary Artery, Am Heart J. 85, pp. 635-647 (1973);Douglas, 1985; Dray et. al. (1993).

[0010] In addition, prostaglandins also are known to cause pain andinflammation. Cyclooxygenase inhibitors, e.g., ibuprofen, are commonlyused in non-surgical and post-operative settings to block the productionof prostaglandins, thereby reducing prostaglandin-mediated pain andinflammation. Flower, R. J., et. al., Analgesic-Antipyretics andAnti-Inflammatory Agents; Drugs Employed in the Treatment of Gout, inGoodman, L. S., et. al., ed., The Pharmacological Basis of Therapeutics,MacMillan Publishing Company, New York, pp. 674-715 (1985).Cyclooxygenase inhibitors are associated with some adverse systemic sideeffects when applied conventionally. For example, indomethacin orketorolac have well recognized gastrointestinal and renal adverse sideeffects.

[0011] As discussed, 5-HT, histamine, bradykinin and prostaglandinscause pain and inflammation. The various receptors through which theseagents mediate their effects on peripheral tissues have been knownand/or debated for the past two decades. Most studies have beenperformed in rats or other animal models. However, there are differencesin pharmacology and receptor sequences between human and animal species.There have been no studies conclusively demonstrating the importance of5-HT, bradykinin or histamine in producing postoperative pain in humans.

[0012] Furthermore, antagonists of these mediators currently are notused for postoperative pain treatment. A class of drugs, termed 5-HT andnorepinephrine uptake antagonists, which includes amitriptyline, hasbeen used orally with moderate success for chronic pain conditions.However, the mechanisms of chronic versus acute pain states are thoughtto be considerably different. In fact, two studies in the acute painsetting using amitriptyline perioperatively have shown no pain-relievingeffect of amitriptyline. Levine, J. D., et. al., Desipramine EnhancesOpiate Postoperative Analgesia, Pain 27, pp. 45-49 (1986); Kerrick, J.M., et. al., Low-Dose Amitriptyline as an Adjunct to Opioids forPostoperative Orthopedic Pain: a Placebo-Controlled Trial Period, Pain52, pp. 325-30 (1993). In both studies the drug was given orally. Thesecond study noted that oral amitriptyline actually produced a loweroverall sense of well-being in postoperative patients, which may be dueto the drug's affinity for multiple amine receptors in the brain.

[0013] Amitriptyline, in addition to blocking the uptake of 5-HT andnorepinephrine, is a potent 5-HT receptor antagonist. Therefore, thelack of efficacy in reducing postoperative pain in thepreviously-mentioned studies would appear to conflict with the proposalof a role for endogenous 5-HT in acute pain. There are a number ofreasons for the lack of acute pain relief found with amitriptyline inthese two studies. (1) The first study (Levine et al., 1986) usedamitriptyline preoperatively for one week up until the night prior tosurgery whereas the second study (Kerrick et al., 1993) only usedamitriptyline postoperatively. Therefore, no amitriptyline was presentin the operative site tissues during the actual tissue injury phase, thetime at which 5-HT is purported to be released. (2) Amitriptyline isknown to be extensively metabolized by the liver. With oraladministration, the concentration of amitriptyline in the operative sitetissues may not have been sufficiently high for a long enough timeperiod to inhibit the activity of postoperatively released 5-HT in thesecond study. (3) Since multiple inflammatory mediators exist, andstudies have demonstrated synergism between the inflammatory mediators,blocking only one agent (5-HT) may not sufficiently inhibit theinflammatory response to tissue injury.

[0014] There have been a few studies demonstrating the ability ofextremely high concentrations (1% -3% solutions—i.e., 10-30 mg permilliliter) of histamine, (H₁) receptor antagonists to act as localanesthetics for surgical procedures. This anesthetic effect is notbelieved to be mediated via H₁ receptors but, rather, due to anon-specific interaction with neuronal membrane sodium channels (similarto the action of lidocaine). Given the side effects (e.g., sedation)associated with these high “anesthetic” concentrations of histaminereceptor antagonists, local administration of histamine receptorantagonists currently is not used in the perioperative setting.

III. SUMMARY OF THE INVENTION

[0015] The present invention provides a solution constituting a mixtureof multiple agents in low concentrations directed at inhibiting locallythe mediators of pain, inflammation, spasm and restenosis in aphysiologic electrolyte carrier fluid. The invention also provides amethod for perioperative delivery of the irrigation solution containingthese agents directly to a surgical site, where it works locally at thereceptor and enzyme levels to preemptively limit pain, inflammation,spasm and restenosis at the site. Due to the local perioperativedelivery method of the present invention, a desired therapeutic effectcan be achieved with lower doses of agents than are necessary whenemploying other methods of delivery (i.e., intravenous, intramuscular,subcutaneous and oral). The anti-pain/anti-inflammation agents in thesolution include agents selected from the following classes of receptorantagonists and agonists and enzyme activators and inhibitors, eachclass acting through a differing molecular mechanism of action for painand inflammation inhibition: (1) serotonin receptor antagonists; (2)serotonin receptor agonists; (3) histamine receptor antagonists; (4)bradykinin receptor antagonists; (5) kallikrein inhibitors; (6)tachykinin receptor antagonists, including neurokinin, and neurokinin₂receptor subtype antagonists; (7) calcitonin gene-related peptide (CGRP)receptor antagonists; (8) interleukin receptor antagonists; (9)inhibitors of enzymes active in the synthetic pathway for arachidonicacid metabolites, including (a) phospholipase inhibitors, including PLA₂isoform inhibitors and PLC_(γ) isoform inhibitors, (b) cyclooxygenaseinhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptorantagonists including eicosanoid EP-1 and EP-4 receptor subtypeantagonists and thromboxane receptor subtype antagonists; (11)leukotriene receptor antagonists including leukotriene B₄ receptorsubtype antagonists and leukotriene D₄ receptor subtype antagonists;(12) opioid receptor agonists, including μ-opioid, δ-opioid, andκ-opioid receptor subtype agonists; (13) purinoceptor agonists andantagonists including P_(2X) receptor antagonists and P_(2Y) receptoragonists; and (14) adenosine triphosphate (ATP)-sensitive potassiumchannel openers. Each of the above agents functions either as ananti-inflammatory agent and/or as an anti-nociceptive, i.e., anti-painor analgesic, agent. The selection of agents from these classes ofcompounds is tailored for the particular application.

[0016] Several preferred embodiments of the solution of the presentinvention also include anti-spasm agents for particular applications.For example, anti-spasm agents may be included alone or in combinationwith anti-pain/anti-inflammation agents in solutions used for vascularprocedures to limit vasospasm, and anti-spasm agents may be included forurologic procedures to limit spasm in the urinary tract and bladderwall. For such applications, anti-spasm agents are utilized in thesolution. For example, an anti-pain/anti-inflammation agent which alsoserves as an anti-spasm agent may be included. Suitableanti-inflammatory/anti-pain agents which also act as anti-spasm agentsinclude serotonin receptor antagonists, tachykinin receptor antagonists,and ATP-sensitive potassium channel openers. Other agents which may beutilized in the solution specifically for their anti-spasm propertiesinclude calcium channel antagonists, endothelin receptor antagonists andthe nitric oxide donors (enzyme activators).

[0017] Specific preferred embodiments of the solution of the presentinvention for use in cardiovascular and general vascular proceduresinclude anti-restenosis agents, which most preferably are used incombination with anti-spasm agents. Suitable anti-restenosis agentsinclude: (1) antiplatelet agents including: (a) thrombin inhibitors andreceptor antagonists, (b) adenosine disphosphate (ADP) receptorantagonists (also known as purinoceptor₁ receptor antagonists), (c)thromboxane inhibitors and receptor antagonists and (d) plateletmembrane glycoprotein receptor antagonists; (2) inhibitors of celladhesion molecules, including (a) selectin inhibitors and (b) integrininhibitors; (3) anti-chemotactic agents; (4) interleukin receptorantagonists (which also serve as anti-pain/anti-inflammation agents);and (5) intracellular signaling inhibitors including: (a) protein kinaseC (PKC) inhibitors and protein tyrosine kinase inhibitors, (b)modulators of intracellular protein tyrosine phosphatases, (c)inhibitors of src homology₂ (SH2) domains, and (d) calcium channelantagonists. Such agents are useful in preventing restenosis of arteriestreated by angioplasty, rotational atherectomy or other cardiovascularor general vascular therapeutic or diagnostic procedure.

[0018] The present invention also provides a method for manufacturing amedicament compounded as a dilute irrigation solution for use incontinuously irrigating an operative site or wound during an operativeprocedure. The method entails dissolving in a physiologic electrolytecarrier fluid a plurality of anti-pain/anti-inflammatory agents, and forsome applications anti-spasm agents and/or anti-restenosis agents, eachagent included at a concentration of preferably no more than 100,000nanomolar, and more preferably no more than 10,000 nanomolar.

[0019] The method of the present invention provides for the delivery ofa dilute combination of multiple receptor antagonists and agonists andenzyme inhibitors and activators directly to a wound or operative site,during therapeutic or diagnostic procedures for the inhibition of pain,inflammation, spasm and restenosis. Since the active ingredients in thesolution are being locally applied directly to the operative tissues ina continuous fashion, the drugs may be used efficaciously at extremelylow doses relative to those doses required for therapeutic effect whenthe same drugs are delivered orally, intramuscularly, subcutaneously orintravenously. As used herein, the term “local” encompasses applicationof a drug in and around a wound or other operative site, and excludesoral, subcutaneous, intravenous and intramuscular administration. Theterm “continuous” as used herein encompasses uninterrupted application,repeated application at frequent intervals (e.g., repeated intravascularboluses at frequent intervals intraprocedurally), and applications whichare uninterrupted except for brief cessations such as to permit theintroduction of other drugs or agents or procedural equipment, such thata substantially constant predetermined concentration is maintainedlocally at the wound or operative site.

[0020] The advantages of low dose applications of agents are three-fold.The most important is the absence of systemic side effects which oftenlimit the usefulness of these agents. Additionally, the agents selectedfor particular applications in the solutions of the present inventionare highly specific with regard to the mediators on which they work.This specificity is maintained by the low dosages utilized. Finally, thecost of these active agents per operative procedure is low.

[0021] The advantages of local administration of the agents via luminalirrigation or other fluid application are the following: (1) localadministration guarantees a known concentration at the target site,regardless of interpatient variability in metabolism, blood flow, etc.;(2) because of the direct mode of delivery, a therapeutic concentrationis obtained instantaneously and, thus, improved dosage control isprovided; and (3) local administration of the active agents directly toa wound or operative site also substantially reduces degradation of theagents through extracellular processes, e.g., first- and second-passmetabolism, that would otherwise occur if the agents were given orally,intravenously, subcutaneously or intramuscularly. This is particularlytrue for those active agents that are peptides, which are metabolizedrapidly. Thus, local administration permits the use of compounds oragents which otherwise could not be employed therapeutically. Forexample, some agents in the following classes are peptidic: bradykininreceptor antagonists; tachykinin receptor antagonists; opioid receptoragonists; CGRP receptor antagonists; and interleukin receptorantagonists. Local, continuous delivery to the wound or operative siteminimizes drug degradation or metabolism while also providing for thecontinuous replacement of that portion of the agent that may bedegraded, to ensure that a local therapeutic concentration, sufficientto maintain receptor, occupancy, is maintained throughout the durationof the operative procedure.

[0022] Local administration of the solution perioperatively throughout asurgical procedure in accordance with the present invention produces apreemptive analgesic, anti-inflammatory, anti-spasmodic oranti-restenotic effect. As used herein, the term “perioperative”encompasses application intraprocedurally, pre- and intraprocedurally,intra- and postprocedurally, and pre-, intra- and postprocedurally. Tomaximize the preemptive anti-inflammatory, analgesic (for certainapplications), antispasmodic (for certain applications) andantirestenotic (for certain applications) effects, the solutions of thepresent invention are most preferably applied pre-, intra- andpostoperatively. By occupying the target receptors or inactivating oractivating targeted enzymes prior to the initiation of significantoperative trauma locally, the agents of the present solution modulatespecific pathways to preemptively inhibit the targeted pathologicprocess. If inflammatory mediators and processes are preemptivelyinhibited in accordance with the present invention before they can exerttissue damage, the benefit is more substantial than if given after thedamage has been initiated.

[0023] Inhibiting more than one inflammatory, spasm or restenosismediator by application of the multiple agent solution of the presentinvention has been shown to dramatically reduce the degree ofinflammation, pain, and spasm, and theoretically should reducerestenosis. The irrigation solutions of the present invention includecombinations of drugs, each solution acting on multiple receptors orenzymes. The drug agents are thus simultaneously effective against acombination of pathologic processes, including pain and inflammation,vasospasm, smooth muscle spasm and restenosis. The action of theseagents is considered to be synergistic, in that the multiple receptorantagonists and inhibitory agonists of the present invention provide adisproportionately increased efficacy in combination relative to theefficacy of the individual agents. The synergistic action of several ofthe agents of the present invention are discussed, by way of example,below in the detailed descriptions of those agents.

[0024] In addition to arthroscopy, the solution of the present inventionmay also be applied locally to any human body cavity or passage,operative wound, traumatic wound (e.g., bums) or in anyoperative/interventional procedure in which irrigation can be performed.These procedures include, but are not limited to, urological procedures,cardiovascular and general vascular diagnostic and therapeuticprocedures, endoscopic procedures and oral, dental and periodontalprocedures. As used hereafter, the term “wound”, unless otherwisespecified, is intended to include surgical wounds,operative/interventional sites, traumatic wounds and bums.

[0025] Used perioperatively, the solution should result in a clinicallysignificant decrease in operative site pain and inflammation relative tocurrently-used irrigation fluids, thereby decreasing the patient'spostoperative analgesic (i.e., opiate) requirement and, whereappropriate, allowing earlier patient mobilization of the operativesite. No extra effort on the part of the surgeon and operating roompersonnel is required to use the present solution relative toconventional irrigation fluids.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention will now be described in greater detail, byway of example, with reference to the accompanying drawings in which:

[0027]FIG. 1 provides a schematic overview of a generic vascular cellshowing molecular targets and flow of signaling information leading tocontraction, secretion and/or proliferation. The integration ofextrinsic signals through receptors, ion channels and other membraneproteins are common to platelets, neutrophils, endothelial cells andsmooth muscle cells. Representative examples of molecular targets areincluded for major groups of molecules which are therapeutic targets ofdrugs included in the solutions of the present invention.

[0028]FIG. 2 provides a detailed diagram of the signaling pathwaysillustrating “crosstalk” between G-protein coupled receptor (GPCR)pathways and receptor tyrosine kinase (RTK) pathways in a vascularsmooth muscle cell. Only representative proteins in each pathway havebeen shown to simplify the flow of information. Activation of GPCRsleads to increases in intracellular calcium and increased protein kinaseC (PKC) activity and subsequent smooth muscle contraction or spasm. Inaddition, “crosstalk” to the RTK signaling pathway occurs throughactivation of PYK2 (a newly discovered protein tyrosine kinase) andPTK-X (an undefined protein tyrosine kinase), triggering proliferation.Conversely, while activation of RTKs directly initiates proliferation,“crosstalk” to the GPCR pathway occurs at the level of PKC activity andcalcium levels. LGR designates ligand-gated receptor, and MAPKdesignates mitogen-activated protein kinase. These interactions definethe basis for synergistic interactions between molecular targetsmediating spasm and restenosis. The GPCR signaling pathway also mediatessignal transduction (FIGS. 3 and 7) leading to pain transmission inother cell types (e.g., neurons).

[0029]FIG. 3 provides a diagram of the G-Protein Coupled Receptor (GPCR)pathway. Specific molecular sites of action for some drugs in apreferred arthroscopic solution of the present invention are identified.

[0030]FIG. 4 provides a diagram of the G-Protein Coupled Receptor (GPCR)pathway including the signaling proteins responsible for ““crosstalk””with the Growth Factor Receptor signaling pathway. Specific molecularsites of action for some drugs in a preferred cardiovascular and generalvascular solution of the present invention are identified. (See alsoFIG. 5).

[0031]FIG. 5 provides a diagram of the Growth Factor Receptor signalingpathway including the signajing proteins responsible for ““crosstalk””with the G-Protein Coupled Receptor signaling pathway. Specificmolecular sites of action for some drugs in a preferred cardiovascularand general vascular solution of the present invention are identified.(See also FIG. 4).

[0032]FIG. 6 provides a diagram of the G-Protein Coupled Receptorpathway including the signaling proteins responsible for ““crosstalk””with the Growth Factor Receptor signaling pathway. Specific molecularsites of action for some drugs in a preferred urologic solution areidentified.

[0033]FIG. 7 provides a diagram of the G-Protein Coupled Receptorpathway. Specific molecular sites of action for some drugs in apreferred general surgical wound solution of the present invention areidentified.

[0034]FIG. 8 provides a diagram of the mechanism of action of nitricoxide (NO) donor drugs and NO causing relaxation of vascular smoothmuscle. Physiologically, certain hormones and transmitters can activatea form of NO synthase in the endothelial cell through elevatedintracellular calcium resulting in increased synthesis of NO. NO donorsmay generate NO extracellularly or be metabolized to NO within thesmooth muscle cell. Extracellular NO can diffuse aacross the endothelialcell or directly enter the smooth muscle cell. The primary target of NOis the soluble guanylate cyclase (GC), leading to activation of acGMP-dependent protein kinase (PKG) and subsequent extrusion of calciumfrom the smooth muscle cell via a membrane pump. NO also hyperpolarizesthe cell by opening potassium channels which in turn cause closure ofvoltage-sensitive calcium channels. Thus, the synergistic interactionsof calcium channel antagonists, potassium channel openers and NO donorsare evident from the above signal transduction pathway.

[0035]FIGS. 9, 10A and 10B provide charts of the percent ofvasoconstriction versus time in control arteries, in the proximalsegment of subject arteries, and in the distal segment of subjectarteries, respectively, for the animal study described in EXAMPLE VIIherein demonstrating the effect on vasoconstriction of infusion withhistamine and serotonin antagonists, used in the solutions of thepresent invention, during balloon angioplasty. FIGS. 11 and 12 providecharts of plasma extravasation versus dosage of amitriptyline, used inthe solutions of the present invention, delivered intravenously andintra-articularly, respectively, to knee joints in which extravasationhas been induced by introduction of 5-hydroxytryptamine in the animalstudy described in EXAMPLE VIII herein.

[0036]FIGS. 13, 14 and 15 provide charts of mean vasoconstriction(negative values) or vasodilation (positive values), ±1 standard errorof the mean for the proximal (FIG. 13), mid (FIG. 14) and distal (FIG.15) segments of arteries treated with saline (N=4) or with a solutionformulated in accordance with the present invention (N=7), at theimmediate and 15 minute post-rotational atherectomy time points in theanimal study of Example XIII described herein.

V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0037] The irrigation solution of the present invention is a dilutesolution of multiple pain/inflammation inhibitory agents, anti-spasmagents and anti-restenosis agents in a physiologic carrier. The carrieris a liquid, which as used herein is intended to encompass biocompatiblesolvents, suspensions, polymerizable and non-polymerizable gels, pastesand salves. Preferably the carrier is an aqueous solution which mayinclude physiologic electrolytes, such as normal saline or lactatedRinger's solution.

[0038] The anti-inflammation/anti-pain agents are selected from thegroup consisting of: (1) serotonin receptor antagonists; (2) serotoninreceptor agonists; (3) histamine receptor antagonists; (4) bradykininreceptor antagonists; (5) kallikrein inhibitors; (6) tachykinin receptorantagonists, including neurokinin, and neurokinin₂ receptor subtypeantagonists; (7) calcitonin gene-related peptide (CGRP) receptorantagonists; (8) interleukin receptor antagonists; (9) inhibitors ofenzymes active in the synthetic pathway for arachidonic acidmetabolites, including (a) phospholipase inhibitors, including PLA₂isoformn inhibitors and PLC₇ isoform inhibitors (b) cyclooxygenaseinhibitors, and (c) lipooxygenase inhibitors; (10) prostanoid receptorantagonists including eicosanoid EP-1 and EP-4 receptor subtypeantagonists and thromboxane receptor subtype antagonists; (11)leukotriene receptor antagonists including leukotriene B4 receptorsubtype antagonists and leukotriene D₄ receptor subtype antagonists;(12) opioid receptor agonists, including μ-opioid, δ-opioid, andκ-opioid receptor subtype agonists; (13) purinoceptor agonists andantagonists including P2X receptor antagonists and P_(2Y) receptoragonists; and (14) adenosine triphosphate (ATP)-sensitive potassiumchannel openers.

[0039] Suitable anti-inflammatory/anti-pain agents which also act asanti-spasm agents include serotonin receptor antagonists, tachykininreceptor antagonists, ATP-sensitive potassium channel openers andcalcium channel antagonists. Other agents which may be utilized in thesolution specifically for their anti-spasm properties includingendothelin receptor antagonists, calcium channel antagonists and thenitric oxide donors (enzyme activators).

[0040] Specific preferred embodiments of the solution of the presentinvention for use in cardiovascular and general vascular proceduresinclude anti-restenosis agents, which most preferably are used incombination with anti-spasm agents. Suitable anti-restenosis agentsinclude: (1) antiplatelet agents including: (a) thrombin inhibitors andreceptor antagonists, (b) adenosine disphosphate (ADP) receptorantagonists (also known as purinoceptor₁ receptor antagonists), (c)thromboxane inhibitors and receptor antagonists and (d) plateletmembrane glycoprotein receptor antagonists; (2) inhibitors of celladhesion molecules, including (a) selectin inhibitors and (b) integrininhibitors; (3) anti-chemotactic agents; (4) interleukin receptorantagonists (which also serve as anti-pain/anti-inflammation agents);and (5) intracellular signaling inhibitors including: (a) protein kinaseC (PKC) inhibitors and protein tyrosine phosphatases, (b) modulators ofintracellular protein tyrosine kinase inhibitors, (c) inhibitors of srchomology₂ (SH2) domains, and (d) calcium channel antagonists. Suchagents are useful in preventing restenosis of arteries treated byangioplasty, rotational atherectomy or other cardiovascular or generalvascular therapeutic procedure.

[0041] In each of the surgical solutions of the present invention, theagents are included in low concentrations and are delivered locally inlow doses relative to concentrations and doses required withconventional methods of drug administration to achieve the desiredtherapeutic effect. It is impossible to obtain an equivalent therapeuticeffect by delivering similarly dosed agents via other (i.e.,intravenous, subcutaneous, intramuscular or oral) routes of drugadministration since drugs given systemically are subject to first- andsecond-pass metabolism. The concentration of each agent is determined inpart based on its dissociation constant, K_(d). As used herein, the termdissociation constant is intended to encompass both the equilibriumdissociation constant for its respective agonist-receptor orantagonist-receptor interaction and the equilibrium inhibitory constantfor its respective activator-enzyme or inhibitor-enzyme interaction.Each agent is preferably included at a low concentration of 0.1 to10,000 times K_(d) nanomolar, except for cyclooxygenase inhibitors,which may be required at larger concentrations depending on theparticular inhibitor selected. Preferably, each agent is included at aconcentration of 1.0 to 1,000 times K_(d) nanomolar and most preferablyat approximately 100 times K_(d) nanomolar. These concentrations areadjusted as needed to account for dilution in the absence of metabolictransformation at the local delivery site. The exact agents selected foruse in the solution, and the concentration of the agents, varies inaccordance with the particular application, as described below.

[0042] A solution in accordance with the present invention can includejust a single or multiple pain/inflammation inhibitory agent(s), asingle or multiple anti-spasm agent(s), a combination of both anti-spasmand pain/inflammation inhibitory agents, or anti-restenosis agents fromthe enumerated classes, at low concentration. However, due to theaforementioned synergistic effect of multiple agents, and the desire tobroadly block pain and inflammation, spasm and restenosis, it ispreferred that multiple agents be utilized.

[0043] The surgical solutions constitute a novel therapeutic approach bycombining multiple pharmacologic agents acting at distinct receptor andenzyme molecular targets. To date, pharmacologic strategies have focusedon the development of highly specific drugs that are selective forindividual receptor subtypes and enzyme isoforms that mediate responsesto individual signaling neurotransmitters and hormones. As an example,endothelin peptides are some of the most potent vasoconstrictors known.Selective antagonists that are specific for subtypes of endothelin (ET)receptors are being sought by several pharmaceutical companies for usein the treatment of numerous disorders involving elevated endothelinlevels in the body. Recognizing the potential role of the receptorsubtype ET_(A) in hypertension, these drug companies specifically aretargeting the development of selective antagonists to the ET_(A)receptor subtype for the anticipated treatment of coronary vasospasm.This standard pharmacologic strategy, although well accepted, is notoptimal since many other vasoconstrictor agents (e.g., serotonin,prostaglandin, eicosanoid, etc.) simultaneously may be responsible forinitiating and maintaining a vasospastic episode (see FIGS. 2 and 4).Furthermore, despite inactivation of a single receptor subtype orenzyme, activation of other receptor subtypes or enzymes and theresultant signal transmission often can trigger a cascade effect. Thisexplains the significant difficulty in employing a singlereceptor-specific drug to block a pathophysiologic process in whichmultiple transmitters play a role. Therefore, targeting only a specificindividual receptor subtype, such as ET_(A), is likely to beineffective.

[0044] In contrast to the standard approach to pharmacologic therapy,the therapeutic approach of the present surgical solutions is based onthe rationale that a combination of drugs acting simultaneously ondistinct molecular targets is required to inhibit the full spectrum ofevents that underlie the development of a pathophysiologic state.Furthermore, instead of targeting a specific receptor subtype alone, thesurgical solutions are composed of drugs that target common molecularmechanisms operating in different cellular physiologic processesinvolved in the development of pain, inflammation, vasospasm, smoothmuscle spasm and restenosis (see FIG. 1). In this way, the cascading ofadditional receptors and enzymes in the nociceptive, inflammatory,spasmodic and restenotic pathways is minimized by the surgicalsolutions. In these pathophysiologic pathways, the surgical solutionsinhibit the cascade effect both “upstream” and “downstream”.

[0045] An example of “upstream” inhibition is the cyclooxygenaseantagonists in the setting of pain and inflammation. The cyclooxygenaseenzymes (COX₁ and COX₂) catalyze the conversion of arachidonic acid toprostaglandin H which is an intermediate in the biosynthesis ofinflammatory and nociceptive mediators including prostaglandins,leukotrienes, and thromboxanes. The cyclooxygenase inhibitors block“upstream” the formation of these inflammatory and nociceptivemediators. This strategy precludes the need to block the interactions ofthe seven described subtypes of prostanoid receptors with their naturalligands. A similar “upstream” inhibitor included in the surgicalsolutions is aprotinin, a kallikrein inhibitor. The enzyme kallikrein, aserine protease, cleaves the high molecular weight kininogens in plasmato produce bradykinins, important mediators of pain and inflammation. Byinhibition of kallikrein, aprotinin effectively inhibits the synthesisof bradykinins, thereby providing an effective “upstream” inhibition ofthese inflammatory mediators.

[0046] The surgical solutions also make use of “downstream” inhibitorsto control the pathophysiologic pathways. In vascular smooth musclepreparations that have been precontracted with a variety ofneurotransmitters (e.g., serotonin, histamine, endothelin, andthromboxane) implicated in coronary vasospasm, ATP-sensitive potassiumchannel openers (KCOs) produce smooth muscle relaxation which isconcentration dependent (Quast et al., 1994; Kashiwabara et al., 1994).The KCOs, therefore, provide a significant advantage to the surgicalsolutions in the settings of vasospasm and smooth muscle spasm byproviding “downstream” antispasmodic effects that are independent of thephysiologic combination of agonists initiating the spasmodic event (seeFIGS. 2 and 4). Similarly, NO donors and voltage-gated calcium channelantagonists can limit vasospasm and smooth muscle spasm initiated bymultiple mediators known to act earlier in the spasmodic pathway.

[0047] The following is a description of suitable drugs falling in theaforementioned classes of anti-inflammation/anti-pain agents, as well assuitable concentrations for use in solutions, of the present invention.While not wishing to be limited by theory, the justification behind theselection of the various classes of agents which is believed to renderthe agents operative is also set forth.

[0048] A. Serotonin Receptor Antagonists

[0049] Serotonin (5-HT) is thought to produce pain by stimulatingserotonin₂ (5-HT₂) and/or serotonin₃ (5-HT₃) receptors on nociceptiveneurons in the periphery. Most researchers agree that 5-HT₃ receptors onperipheral nociceptors mediate the immediate pain sensation produced by5-HT (Richardson et al., 1985). In addition to inhibiting 5-HT-inducedpain, 5-HT₃ receptor antagonists, by inhibiting nociceptor activation,also may inhibit neurogenic inflammation. Barnes P. J., et. al.,Modulation of Neurogenic Inflammation: Novel Approaches to InflammatoryDisease, Trends in Pharmacological Sciences 11, pp. 185-189 (1990). Astudy in rat ankle joints, however, claims the 5-HT₂ receptor isresponsible for nociceptor activation by 5-HT. Grubb, B. D., et. al., AStudy of 5-HT-Receptors Associated with Afferent Nerves Located inNormal and Inflamed Rat Ankle Joints, Agents Actions 25, pp.216-18(1988). Therefore, activation of 5-HT₂ receptors also may play a role inperipheral pain and neurogenic inflammation.

[0050] One goal of the solution of the present invention is to blockpain and a multitude of inflammatory processes. Thus, 5-HT₂ and 5-HT₃receptor antagonists are both suitably used, either individually ortogether, in the solution of the present invention, as shall bedescribed subsequently. Amitriptyline (Elavil™) is a suitable 5-HT₂receptor antagonist for use in the present invention. Amitriptyline hasbeen used clinically for numerous years as an anti-depressant, and isfound to have beneficial effects in certain chronic pain patients.Metoclopramide (Reglan™) is used clinically as an anti-emetic drug, butdisplays moderate affinity for the 5-HT₃ receptor and can inhibit theactions of 5-HT at this receptor, possibly inhibiting the pain due to5-HT release from platelets. Thus, it also is suitable for use in thepresent invention.

[0051] Other suitable 5-HT₂ receptor antagonists include imipramine,trazodone, desipramine and ketanserin. Ketanserin has been usedclinically for its anti-hypertensive effects. Hedner, T., et. al.,Effects of a New Serotonin Antagonist, Ketanserin, in Experimental andClinical Hypertension, Am J of Hypertension, pp. 317s-23s (Jul. 1988).Other suitable 5-HT₃ receptor antagonists include cisapride andondansetron. The cardiovascular and general vascular solution also maycontain a serotonin_(1B) (also known as serotonin_(1D) _(β) ) antagonistbecause serotonin has been shown to produce significant vascular spasmvia activation of the serotonin_(1B) receptors in humans. Kaumann, A.J., et al., Variable Participation of 5-HT1-Like Receptors and 5-IT2Receptors in Serotonin-Induced Contraction of Human Isolated CoronaryArteries, Circulation 90, pp. 1141-53 (1994). Suitable serotonin_(1B)receptor antagonists include yohimbine,N-[-methoxy-3-(4-methyl-1-piperanzinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide(“GR127935”) and methiothepin. Therapeutic and preferred concentrationsfor use of these drugs in the solution of the present invention are setforth in Table 1. TABLE 1 Therapeutic and Preferred Concentrations ofPain/Inflammation Inhibitory Agents Therapeutic Preferred ConcentrationsConcentrations Class of Agent (Nanomolar) (Nanomolar) Serotonin₂Receptor Antagonists: amitriptyline 0.1-1,000 50-500 imipramine0.1-1,000 50-500 trazodone 0.1-2,000 50-500 desipramine 0.1-1,000 50-500ketanserin 0.1-1,000 50-500 Serotonin₃ Receptor Antagonists: tropisetron0.01-100   0.05-50   metoclopramide   10-10,000  200-2,000 cisapride0.1-1,000 20-200 ondansetron 0.1-1,000 20-200 Serotonin_(1B) (Human1D_(β)) Antagonists: yohimbine 0.1-1,000 50-500 GR127935 0.1-1,00010-500 methiothepin 0.1-500    1-100

[0052] B. Serotonin Receptor Agonists

[0053] 5-HT_(1A), 5-HT_(1B) and 5-HT_(1D) receptors are known to inhibitadenylate cyclase activity. Thus including a low dose of theseserotonin_(1A), serotonin_(1B) and serotonin_(1D) receptor agonists inthe solution should inhibit neurons mediating pain and inflammation. Thesame action is expected from serotonin_(1E) and serotonin_(1F) receptoragonists because these receptors also inhibit adenylate cyclase.

[0054] Buspirone is a suitable 1A receptor agonist for use in thepresent invention. Sumatriptan is a suitable 1A, 1IB, 1D and 1F receptoragonist. A suitable 1B and 1D receptor agonist is dihydroergotamine. Asuitable IE receptor agonist is ergonovine. Therapeutic and preferredconcentrations for these receptor agonists are provided in Table 2.TABLE 2 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) Serotonin_(1A) Agonists:buspirone 1-1,000 10-200 sumatriptan 1-1,000 10-200 Serotonin_(1B)Agonists: dihydroergotamine 0.1-1,000   10-100 sumatriptan 1-1,00010-200 Serotonin_(1D) Agonists: dihydroergotamine 0.1-1,000   10-100sumatriptan 1-1,000 10-200 Serotonin_(1E) Agonists: ergonovine 10-2,000  100-1,000 Serotonin_(1F) Agonists: sumatriptan 1-1,000 10-200

[0055] C. Histamine Receptor Antagonists

[0056] Histamine receptors generally are divided into histamine₁ (H₁)and histamine₂ (H₂) subtypes. The classic inflammatory response to theperipheral administration of histamine is mediated via the H₁ receptor.Douglas, 1985. Therefore, the solution of the present inventionpreferably includes a histamine H₁ receptor antagonist. Promethazine(Phenergan™) is a commonly used anti-emetic drug which potently blocksH₁ receptors, and is suitable for use in the present invention.Interestingly, this drug also has been shown to possess local anestheticeffects but the concentrations necessary for this effect are severalorders higher than that necessary to block H₁ receptors, thus, theeffects are believed to occur by different mechanisms. The histaminereceptor antagonist concentration in the solution is sufficient toinhibit H₁ receptors involved in nociceptor activation, but not toachieve a “local anesthetic” effect, thereby eliminating the concernregarding systemic side effects.

[0057] Histamine receptors also are known to mediate vasomotor tone inthe coronary arteries. In vitro studies in the human heart havedemonstrated that the histamine₁ receptor subtype mediates contractionof coronary smooth muscle. Ginsburg, R., et al., Histamine Provocationof Clinical Coronary Artery Spasm: Implications Concerning Pathogenesisof Variant Angina Pectoris, American Heart J., Vol. 102, pp. 819-822,(1980). Some studies suggest that histamine-induced hypercontractilityin the human coronary system is most pronounced in the proximal arteriesin the setting of atherosclerosis and the associated denudation of thearterial endothelium. Keitoku, M. et al., Different Histamine Actions inProximal and Distal Human Coronary Arteries in Vitro, CardiovascularResearch 24, pp. 614-622, (1990). Therefore, histamine receptorantagonists may be included in the cardiovascular irrigation solution.

[0058] Other suitable H₁ receptor antagonists include terfenadine,diphenhydramine, amitriptyline, mepyramine and tripolidine. Becauseamitriptyline is also effective as a serotonin₂ receptor antagonist, ithas a dual function as used in the present invention. Suitabletherapeutic and preferred concentrations for each of these H₁ receptorantagonists are set forth in Table 3. TABLE 3 Therapeutic and PreferredConcentrations of Pain/Inflammation Inhibitory Agents TherapeuticPreferred Concentrations Concentrations Class of Agent (Nanomolar)(Nanomolar) Histamine₁ Receptor Antagonists: promethazine 0.1-1,00050-200 diphenhydramine 0.1-1,000 50-200 amitriptyline 0.1-1,000 50-500terfenadine 0.1-1,000 50-500 mepyramine (pyrilamine) 0.1-1,000  5-200tripolidine 0.01-100   5-20

[0059] D. Bradykinin Receptor Antagonists

[0060] Bradykinin receptors generally are divided into bradykinin₁ (B₁)and bradykinin₂ (B₂) subtypes. Studies have shown that acute peripheralpain and inflammation produced by bradykinin are mediated by the B₂subtype whereas bradykinin-induced pain in the setting of chronicinflammation is mediated via the B₁ subtype. Perkins, M. N., et. al.,Antinociceptive Activity of the Bradykinin B1 and B2 ReceptorAntagonists, des-Arg ⁹ , [Leu ⁸]-BK and HOE 140, in Two Models ofPersistent Hyperalgesia in the Rat, Pain 53, pp. 191-97 (1993); Dray,A., et. al., Bradykinin and Inflammatory Pain, Trends Neurosci 16, pp.99-104 (1993), each of which references is hereby expressly incorporatedby reference.

[0061] At present, bradykinin receptor antagonists are not usedclinically. These drugs are peptides (small proteins), and thus theycannot be taken orally, because they would be digested. Antagonists toB₂ receptors block bradykinin-induced acute pain and inflammation. Drayet. al., 1993. B₁ receptor antagonists inhibit pain in chronicinflammatory conditions. Perkins et al., 1993; Dray et. al., 1993.Therefore, depending on the application, the solution of the presentinvention preferably includes either or both bradykinin B₁ and B₂receptor antagonists. For example, arthroscopy is performed for bothacute and chronic conditions, and thus an irrigation solution forarthroscopy could include both B₁ and B₂ receptor antagonists.

[0062] Suitable bradykinin receptor antagonists for use in the presentinvention include the following bradykinin, receptor antagonists: the[des-Arg¹⁰] derivative of D-Arg-(Hyp³-Thi⁵-D-Tic⁷-Oic⁸)-BK (“the[des-Arg¹⁰] derivative of HOE 140”, available from HoechstPharmaceuticals); and [Leu8] des-Arg⁹-BK. Suitable bradykinin₂ receptorantagonists include: [D-Phe⁷]-BK; D-Arg-(Hyp³-Thi^(5,8)-D-Phe⁷)-BK (“NPC349”); D-Arg-(Hyp³—D-Phe⁷)-BK (“NPC 567”); andD-Arg-(Hyp³-Thi⁵-D-Tic⁷-Oic⁸)-BK (“HOE 140”). These compounds are morefully described in the previously incorporated Perkins et. al. 1993 andDray et. al. 1993 references. Suitable therapeutic and preferredconcentrations are provided in Table 4. TABLE 4 Therapeutic andPreferred Concentrations of Pain/Inflammation Inhibitory AgentsTherapeutic Preferred Concentrations Concentrations Class of Agent(Nanomolar) (Nanomolar) Bradykinin₁ Receptor Antagonists: [Leu⁸]des-Arg⁹-BK 1-1,000 50-500 [des-Arg¹⁰] derivative of HOE 140 1-1,00050-500 [leu⁹] [des-Arg¹⁰] kalliden 0.1-500    10-200 Bradykinin₂Receptor Antagonists: [D-Phe⁷]-BK 100-10,000   200-5,000 NPC 349 1-1,00050-500 NPC 567 1-1,000 50-500 HOE 140 1-1,000 50-500

[0063] E. Kallikrein Inhibitors

[0064] The peptide bradykinin is an important mediator of pain andinflammation, as noted previously. Bradykinin is produced as. a cleavageproduct by the action of kallikrein on high molecular weight kininogensin plasma. Therefore kallikrein inhibitors are believed to betherapeutic in inhibiting bradykinin production and resultant pain andinflammation. A suitable kallikrein inhibitor for use in the presentinvention is aprotinin. Suitable concentrations for use in the solutionsof the present invention are set forth below in Table 5. TABLE 5Therapeutic and Preferred Concentrations of Pain/Inflammation InhibitoryAgents Therapeutic Preferred Concentrations Concentrations Class ofAgent (Nanomolar) (Nanomolar) Kallikrein Inhibitor: Aprotinin 0.1-1,00050-500

[0065] F. Tachykinin Receptor Antagonists

[0066] Tachykinins (TKs) are a family of structurally related peptidesthat include substance P, neurokinin A (NKA) and neurokinin B (NKB).Neurons are the major source of TKs in the periphery. An importantgeneral effect of TKs is neuronal stimulation, but other effects includeendothelium-dependent vasodilation, plasma protein extravasation, mastcell recruitment and degranulation and stimulation of inflammatorycells. Maggi, C. A., Gen. Pharmacol., Vol. 22, pp. 1-24 (1991). Due tothe above combination of physiological actions mediated by activation ofTK receptors, targeting of TK receptors is a reasonable approach for thepromotion of analgesia and the treatment of neurogenic inflammation.

1. Neurokinin₁ Receptor Subtype Antagonists

[0067] Substance P activates the neurokinin receptor subtype referred toas NK₁. Substance P is an undecapeptide that is present in sensory nerveterminals. Substance P is known to have multiple actions which produceinflammation and pain in the periphery after C-fiber activation,including vasodilation, plasma extravasation and degranulation of mastcells. Levine, J. D., et. al., Peptides and the Primary AfferentNociceptor, J. Neurosci. 13, p. 2273 (1993). A suitable Substance Pantagonist is([D-Pro⁹[spiro-garmma-lactam]Leu¹⁰,Trp¹¹]physalaemin-(1-11)) (“GR82334”). Other suitable antagonists for use in the present inventionwhich act on the NK₁ receptor are: 1-imino-2-(2-methoxy-phenyl)-ethyl)-7,7-diphenyl-4-perhydroisoindolone(3aR,7aR)(“RP 67580”); and2S,3S-cis-3-(2-methoxybenzylaamino)-2-benzhydrylquinuclidine (“CP96,345”). Suitable concentrations for these agents are set forth inTable 6. TABLE 6 Therapeutic and Preferred Concentrations ofPain/Inflammation Inhibitory Agents Therapeutic Preferred ConcentrationsConcentrations Class of Agent (Nanomolar) (Nanomolar) Neurokinin₁Receptor Subtype Antagonists GR 82334 1-1,000 10-500  CP 96,345 1-10,000 100-1,000 RP 67580 0.1-1,000   100-1,000

2. Neurokinin₂ Receptor Subtype Antagonists

[0068] Neurokinin A is a peptide which is colocalized in sensory neuronswith substance P and which also promotes inflammation and pain.Neurokinin A activates the specific neurokinin receptor referred to asNK₂. Edmonds-Alt, S., et. al., A Potent and Selective Non-PeptideAntagonist of the Neurokinin A (NK ₂) Receptor, Life Sci. 50:PL101(1992). In the urinary tract, TKs are powerful spasmogens acting throughonly the NK₂ receptor in the human bladder, as well as the human urethraand ureter. Maggi, C. A., Gen. Pharmacol., Vol. 22, pp. 1-24 (1991).Thus, the desired drugs for inclusion in a surgical solution for use inurological procedures would contain an antagonist to the NK₂ receptor toreduce spasm. Examples of suitable NK₂ antagonists include:((S)-N-methyl-N-[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide(“(±)-SR 48968”); Met-Asp-Trp-Phe-Dap-Leu (“MEN 10,627”); andcyc(Gln-Trp-Phe-Gly-Leu-Met) (“L 659,877”). Suitable concentrations ofthese agents are provided in Table 7. TABLE 7 Therapeutic and PreferredConcentrations of Pain/Inflammation Inhibitory Agents TherapeuticPreferred Concentrations Concentrations Class of Agent (Nanomolar)(Nanomolar) Neurokinin₂ Receptor Subtype Antagonists: MEN 10,627 1-1,00010-1,000 L 659,877 10-10,000 100-10,000 (±)-SR 48968 10-10,000100-10,000

[0069] G. CGRP Receptor Antagonists

[0070] Calcitonin gene-related peptide (CGRP) is a peptide which is alsocolocalized in sensory neurons with substance P, and which acts as avasodilator and potentiates the actions of substance P. Brain, S. D.,et. al., Inflammatory Oedema Induced by Synergism Between CalcitoninGene-Related Peptide (CGRP) and Mediators of Increased VascularPermeability, Br. J. Pharmacol. 99, p. 202 (1985). An example of asuitable CGRP receptor antagonist is α-CGRP-(8-37), a truncated versionof CGRP. This polypeptide inhibits the activation of CGRP receptors.Suitable concentrations for this agent are provided in Table 8. TABLE 8Therapeutic and Preferred Concentrations of Pain/Inflammation InhibitoryAgents Therapeutic Preferred Concentrations Concentrations Class ofAgent (Nanomolar) (Nanomolar) CGRP Receptor Antagonist: α-CGRP-(8-37)1-1,000 10-500

[0071] H. Interleukin Receptor Antagonist

[0072] Interleukins are a family of peptides, classified as cytokines,produced by leukocytes and other cells in response to inflammatorymediators. Interleukins (IL) may be potent hyperalgesic agentsperipherally. Ferriera, S. H., et. al., Interleukin-1β as a PotentHyperalgesic Agent Antagonized by a Tripeptide Analogue, Nature 334, p.698 (1988). An example of a suitable IL-1β receptor antagonist isLys-D-Pro-Thr, which is a truncated version of IL-1β. This tripeptideinhibits the activation of IL-1β receptors. Suitable concentrations forthis agent are provided in Table 9. TABLE 9 Therapeutic and PreferredConcentrations of Pain/Inflammation Inhibitory Agents TherapeuticPreferred Concentrations Concentrations Class of Agent (Nanomolar)(Nanomolar) Interleukin Receptor Antagonist: Lys-D-Pro-Thr 1-1,00010-500

[0073] I. Inhibitors of Enzymes Active in the Synthetic Pathway forArachidonic Acid Metabolites

1. Phospholipase Inhibitors

[0074] The production of arachidonic acid by phospholipase A₂ (PLA₂)results in a cascade of reactions that produces numerous mediators ofinflammation, know as eicosanoids. There are a number of stagesthroughout this pathway that can be inhibited, thereby decreasing theproduction of these inflammatory mediators. Examples of inhibition atthese various stages are given below.

[0075] Inhibition of the enzyme PLA₂ isoform inhibits the release ofarachidonic acid from cell membranes, and therefore inhibits theproduction of prostaglandins and leukotrienes resulting in decreasedinflammation and pain. Glaser, K. B., Regulation of Phospholipase A2Enzymes: Selective Inhibitors and Their Pharmacological Potential, Adv.Pharmacol. 32, p. 31 (1995). An example of a suitable PLA₂ isoforminhibitor is manoalide. Suitable concentrations for this agent areincluded in Table 10. Inhibition of the phospholipase C_(γ) (PLC_(γ))isoform also will result in decreased production of prostanoids andleukotrienes, and, therefore, will result in decreased pain andinflammation. An example of a PLC_(γ) isoform inhibitor is1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione.TABLE 10 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Concentrations ConcentrationsClass of Agent (Nanomolar) (Nanomolar) PLA₂ Isoform Inhibitor: manoalide100-100,000 500-10,000

2. Cyclooxygenase Inhibitors

[0076] Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used asanti-inflammatory, anti-pyretic, anti-thrombotic and analgesic agents.Lewis, R. A., Prostaglandins and Leukotrienes, In: Textbook ofRheumatology, 3d ed. (Kelley W. N., et. al., eds.), p. 258 (1989). Themolecular targets for these drugs are type I and type II cyclooxygenases(COX-1 and COX-2). These enzymes are also known as Prostaglandin HSynthase (PGHS)-1 (constitutive) and -2 (inducible), and catalyze theconversion of arachidonic acid to Prostaglandin H which is anintermediate in the biosynthesis of prostaglandins and thromboxanes. TheCOX-2 enzyme has been identified in endothelial cells, macrophages, andfibroblasts. This enzyme is induced by IL-1 and endotoxin, and itsexpression is upregulated at sites of inflammation. Constitutiveactivity of COX-1 and induced activity of COX-2 both lead to synthesisof prostaglandins which contribute to pain and inflammation.

[0077] NSAIDs currently on the market (diclofenac, naproxen,indomethacin, ibuprofen, etc.) are generally nonselective inhibitors ofboth isoforms of COX, but may show greater selectively for COX-1 overCOX-2, although this ratio varies for the different compounds. Use ofCOX-1 and 2 inhibitors to block formation of prostaglandins represents abetter therapeutic strategy than attempting to block interactions of thenatural ligands with the seven described subtypes of prostanoidreceptors. Reported antagonists of the eicosanoid receptors (EP-1, EP-2,EP-3) are quite rare and only specific, high affinity antagonists of thethromboxane A2 receptor have been reported. Wallace, J. and Cirino, G.Trends in Pharm. Sci., Vol. 15 pp. 405-406 (1994).

[0078] The oral, intravenous or intramuscular use of cyclooxygenaseinhibitors is contraindicated in patients with ulcer disease, gastritisor renal impairment. In the United States, the only available injectableform of this class of drugs is ketorolac (Toradol™), available fromSyntex Pharmaceuticals, which is conventionally used intramuscularly orintravenously in postoperative patients but, again, is contraindicatedfor the above-mentioned categories of patients. The use of ketorolac, orany other cyclooxygenase inhibitor(s), in the solution in substantiallylower dosages than currently used perioperatively may allow the use ofthis drug in otherwise contraindicated patients. The addition of acyclooxygenase inhibitor to the solutions of the present invention addsa distinct mechanism for inhibiting the production of pain andinflammation during arthroscopy or other therapeutic or diagnosticprocedure.

[0079] Preferred cyclooxygenase inhibitors for use in the presentinvention are keterolac and indomethacin. Of these two agents,indomethacin is less preferred because of the relatively high dosagesrequired. Therapeutic and preferred concentrations for use in thesolution are provided in Table 11. TABLE 11 Therapeutic and PreferredConcentrations of Pain/Inflammation Inhibitory Agents TherapeuticPreferred Class of Agent Concentrations Concentrations CyclooxygenaseInhibitors: (Nanomolar) (Nanomolar) ketorolac   100-10,000   500-5,000indomethacin  1,000-500,000  10,000-200,000

3. Lipooxygenase Inhibitors

[0080] Inhibition of the enzyme lipooxygenase inhibits the production ofleukotrienes, such as leukotriene B₄, which is known to be an importantmediator of inflammation and pain. Lewis, R. A., Prostaglandins andLeukotrienes, In: Textbook of Rheumatology, 3d ed. (Kelley W. N., et.al., eds.), p. 258 (1989). An example of a 5-lipooxygenase antagonist is2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (“AA861”), suitable concentrations for which are listed in Table 12. TABLE12 Therapeutic and Preferred Concentrations of Pain/InflammationInhibitory Agents Therapeutic Preferred Class of Agent ConcentrationsConcentrations Lipooxygenase Inhibitor: (Nanomolar) (Nanomolar) AA861100-10,000 500-5,000

[0081] J. Prostanoid Receptor Antagonists

[0082] Specific prostanoids produced as metabolites of arachidonic acidmediate their inflammatory effects through activation of prostanoidreceptors. Examples of classes of specific prostanoid antagonists arethe eicosanoid EP-1 and EP-4 receptor subtype antagonists and thethromboxane receptor subtype antagonists. A suitable prostaglandin E₂receptor antagonist is8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid,2-acetylhydrazide (“SC 19220”). A suitable thromboxane receptor subtypeantagonist is [15-[1α, 2β(5Z), 3β,4α]-7-[3-[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxobicyclo-[2,2,1]-hept-2-yl]-5-heptanoicacid (“SQ 29548”). Suitable concentrations for these agents are setforth in Table 13. TABLE 13 Therapeutic and Preferred Concentrations ofPain/Inflammation Inhibitory Agents Therapeutic Preferred Class of AgentConcentrations Concentrations Bicosanoid EP-1 Antagonist: (Nanomolar)(Nanomolar) SC 19220 100-10,000 500-5,000

[0083] K. Leukotriene Receptor Antagonists

[0084] The leukotrienes (LTB₄, LTC₄, and LTD₄) are products of the5-lipooxygenase pathway of arachidonic acid metabolism that aregenerated enzymatically and have important biological properties.Leukotrienes are implicated in a number of pathological conditionsincluding inflammation. Specific antagonists are currently being soughtby many pharmaceutical companies for potential therapeutic interventionin these pathologies. Halushka, P. V., et al., Annu. Rev. Pharmacol.Toxicol. 29: 213-239 (1989); Ford-Hutchinson, A. Crit. Rev. Immunol. 10:1-12 (1990). The LTB₄ receptor is found in certain immune cellsincluding eosinophils and neutrophils. LTB₄ binding to these receptorsresults in chemotaxis and lysosomal enzyme release thereby contributingto the process of inflammation. The signal transduction processassociated with activation of the LTB₄ receptor involvesG-protein-mediated stimulation of phosphotidylinositol (PI) metabolismand elevation of intracellular calcium (see FIG. 2).

[0085] An example of a suitable leukotriene B₄ receptor antagonist is SC(+)-(S)-7-(3-(2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)-carbonyl]phenoxy(propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoicacid (“SC 53228”). Concentrations for this agent that are suitable forthe practice of the present invention are provided in Table 14. Othersuitable leukotriene B₄ receptor antagonists include[3-[-2(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino-3-oxopropyl)thio]methyl]thiopropanoic acid (“MK0571”) and the drugs LY 66,071 and ICI 20,3219. MK 0571 also acts as aLTD₄ receptor subtype antagonist. TABLE 14 Therapeutic and PreferredConcentrations of Pain/Inflammation Inhibitory Agents TherapeuticPreferred Class of Agent Concentrations Concentrations Leukotriene B₄Antagonist: (Nanomolar) (Nanomolar) SC 53228 100-10,000 500-5,000

[0086] L. Opioid Receptor Agonists

[0087] Activation of opioid receptors results in anti-nociceptiveeffects and, therefore, agonists to these receptors are desirable.Opioid receptors include the μ-, δ- and κ-opioid receptor subtypes. Thei-receptors are located on sensory neuron terminals in the periphery andactivation of these receptors inhibits sensory neuron activity. Basbaum,A. I., et. al., Opiate analgesia: How Central is a Peripheral Target?,N. Engl. J. Med., 325:1168 (1991). δ- and κ-receptors are located onsympathetic efferent terminals and inhibit the release ofprostaglandins, thereby inhibiting pain and inflammation. Taiwo, Y. O.,et. al., Kappa- and Delta-Opioids Block Sympathetically DependentHyperalgesia, J. Neurosci., Vol. 11, page 928 (1991). The opioidreceptor subtypes are members of the G-protein coupled receptorsuperfamily. Therefore, all opioid receptor agonists interact andinitiate signaling through their cognate G-protein coupled receptor (seeFIGS. 3 and 7). Examples of suitable μ-opioid receptor agonists arefentanyl and Try-D-Ala-Gly-[N-MePhe]-NH(CH₂)—OH (“DAMGO”). An example ofa suitable δ-opioid receptor agonist is [D-Pen²,D-Pen⁵]enkephalin(“DPDPE”). An example of a suitable K-opioid receptor agonist is(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidnyl)cyclohexyl]-benzeneacetamide (“U50,488”). Suitable concentrations for each of these agentsare set forth in Table 15. TABLE 15 Therapeutic and PreferredConcentrations of Pain/Inflammation Inhibitory Agents TherapeuticPreferred Concentrations Concentrations Class of Agent (Nanomolar)(Nanomolar) μ-Opioid Agonist: DAMGO 0.1-100 0.5-20  sufentanyl 0.01-50  1-20 fentanyl 0.1-500  10-200 PLO17 0.05-50  0.25-10   δ-OpioidAgonist: DPDPE 0.1-500  1.0-100 κ-Opioid Agonist: U50,488 0.1-500 1.0-100

[0088] M. Purinoceptor Antagonists and Agonists

[0089] Extracellular ATP acts as a signaling molecule throughinteractions with P₂ purinoceptors. One major class of purinoceptors arethe P_(2x) purinoceptors which are ligand-gated ion channels possessingintrinsic ion channels permeable to Na⁺, K⁺, and Ca²⁺. P_(2x) receptorsdescribed in sensory neurons are important for primary afferentneurotransmission and nociception. ATP is known to depolarize sensoryneurons and plays a role in nociceptor activation since ATP releasedfrom damaged cells stimulates P_(2X) receptors leading to depolarizationof nociceptive nerve-fiber terminals. The P2X₃ receptor has a highlyrestricted distribution (Chen, C. C., et. al., Nature, Vol. 377, pp.428-431 (1995)) since it is selectively expressed in sensory C-fibernerves that run into the spinal cord and many of these C-fibers areknown to carry the receptors for painful stimuli. Thus, the highlyrestricted localization of expression for the P2X₃ receptor subunitsmake these subtypes excellent targets for analgesic action (see FIGS. 3and 7).

[0090] Suitable antagonists of P_(2XX)/ATP purinoceptors for use in thepresent invention include, by way of example, suramin andpyridoxylphosphate-6-azophenyl-2,4-disulfonic acid (“PPADS”). Suitableconcentrations for these agents are provided in Table 16.

[0091] Agonists of the P_(2Y) receptor, a G-protein coupled receptor,are known to effect smooth muscle relaxation through elevation ofinositol triphosphate (IP₃) levels with a subsequent increase inintracellular calcium. An example of a P_(2Y) receptor agonist is2-me-S-ATP. TABLE 16 Therapeutic and Preferred Concentrations ofPain/Inflammation Inhibitory Agents Therapeutic Preferred Class of AgentConcentrations Concentrations Purinoceptor Antagonists: (Nanomolar)(Nanomolar) suramin 100-100,000 10,000-100,000 PPADS 100-100,00010,000-100,000

[0092] N. Adenosine Triphosphate (ATP)-Sensitive Potassium ChannelOpeners

[0093] ATP-sensitive potassium channels have been discovered in numeroustissues, including vascular and non-vascular smooth muscle and brain,and binding studies using radiolabeled ligands have confirmed theirexistence. Opening of these channels causes potassium (K⁺) efflux andhyperpolarizes the cell membrane (see FIG. 2). This hyperpolarizationinduces a reduction in intracellular free calcium through inhibition ofvoltage-dependent calcium (Ca²⁺) channels and receptor operated Ca²⁺channels. These combined actions drive the cell (e.g., smooth musclecell) into a relaxed state or one which is more resistant to activationand, in the case of vascular smooth muscle, results in vasorelaxation.K⁺ channel openers (KCOs) have been characterized as having potentantihypertensive activity in vivo and vasorelaxant activity in vitro(see FIG. 4). K⁺ channel openers (KCOs) also have been shown to preventstimulus coupled secretion and are considered to act on prejunctionalneuronal receptors and thus will inhibit effects due to nervestimulation and release of inflammatory mediators. Quast, U., et. al.,Cellular Pharmacology of Potassium Channel Openers in Vascular SmoothMuscle, Cardiovasc. Res., Vol. 28, pp. 805-810 (1994).

[0094] Synergistic interactions between endothelin (ETA) antagonists andopeners of ATP-sensitive potassium channels (KCOs) are expected inachieving vasorelaxation or smooth muscle relaxation. A rationale fordual use is based upon the fact that these drugs have differentmolecular mechanisms of action in promoting relaxation of smooth muscleand prevention of vasospasm. An initial intracellular calcium elevationin smooth muscle cells induced by the ET_(A) receptor subsequentlytriggers activation of voltage-dependent channels and the entry ofextracellular calcium which is required for contraction. Antagonists ofthe ET_(A) receptor will specifically block this receptor mediatedeffect but not block increases in calcium triggered by activation ofother G-protein coupled receptors on the muscle cell.

[0095] Potassium-channel opener drugs, such as pinacidil, will openthese channels causing K⁺ efflux and hyperpolarization of the cellmembrane. This hyperpolarization will act to reduce contraction mediatedby other receptors by the following mechanisms: (1) it will induce areduction in intracellular free calcium through inhibition ofvoltage-dependent Ca²⁺ channels by reducing the probability of openingL-type or T-type calcium channels, (2) it will restrain agonist induced(receptor operated channels) Ca²⁺ release from intracellular sourcesthrough inhibition of inositol triphosphate (IP₃) formation, and (3) itwill lower the efficiency of calcium as an activator of contractileproteins. Consequently, combined actions of these two classes of drugswill clamp the target cells into a relaxed state or one which is moreresistant to activation.

[0096] Suitable ATP-sensitive K⁺ channel openers for the practice of thepresent invention include: (−)pinacidil; cromakalim; nicorandil;minoxidil;N-cyano-N′-[1,1-dimethyl-[2,2,3,3-³H]propyl]-N″-(3-pyridinyl)guanidine(“P 1075”); and N-cyano-N′-(2-nitroxyethyl)-3-pyridinecarboximidamidemonomethansulphonate (“KRN 2391”). Concentrations for these agents areset forth in Table 17. TABLE 17 Therapeutic and Preferred Concentrationsof Pain/Inflammation Inliibitory Agents Therapeutic Preferred Class ofAgent Concentrations Concentrations ATP-Sensitive K^(±) Channel Opener:(Nanomolar) (Nanomolar) cromakalim 10-10,000 100-10,000 nicorandil10-10,000 100-10,000 minoxidil 10-10,000 100-10,000 P 1075 0.1-1,000  10-1,000 KRN 2391  1-10,000 100-1,000  (−)pinacidil  1-10,000 100-1,000 

[0097] I. Anti-Spasm Agents

1. Multifunction Agents

[0098] Several of the anti-pain/anti-inflammatory agents described abovealso serve to inhibit vasoconstriction or smooth muscle spasm. As such,these agents also perform the function of anti-spasm agents, and thusare beneficially used in vascular and urologic applications.Anti-inflammatory/anti-pain agents that also serve as anti-spasm agentsinclude: serotonin receptor antagonists, particularly, serotonin₂antagonists; tachykinin receptor antagonists and ATP-sensitive potassiumchannel openers.

2. Nitric Oxide Donors

[0099] Nitric oxide donors may be included in the solutions of thepresent invention particularly for their anti-spasm activity. Nitricoxide (NO) plays a critical role as a molecular mediator of manyphysiological processes, including vasodilation and regulation of normalvascular tone. Within endothelial cells, an enzyme known as NO synthase(NOS) catalyzes the conversion of L-arginine to NO which acts as adiffusible second messenger and mediates responses in adjacent smoothmuscle cells (see FIG. 8). NO is continuously formed and released by thevascular endothelium under basal conditions which inhibits contractionsand controls basal coronary tone and is produced in the endothelium inresponse to various agonists (such as acetylcholine) and otherendothelium dependent vasodilators. Thus, regulation of NO synthaseactivity and the resultant levels of NO are key molecular targetscontrolling vascular tone (see FIG. 8). Muramatsu, K., et. al., Coron.Artery Dis., Vol. 5, pp. 815-820 (1994).

[0100] Synergistic interactions between NO donors and openers ofATP-sensitive potassium channels (KCOS) are expected to achievevasorelaxation or smooth muscle relaxation. A rationale for dual use isbased upon the fact that these drugs have different molecular mechanismsof action in promoting relaxation of smooth muscle and prevention ofvasospasm. There is evidence from cultured coronary arterial smoothmuscle cells that the vasoconstrictors: vasopressin, angotensin II andendothelin, all inhibit K_(ATP) currents through inhibition of proteinkinase A. In addition, it has been reported that K_(ATP) current inbladder smooth muscle is inhibited by muscarinic agonists. The actionsof NO in mediating smooth muscle relaxation occur via independentmolecular pathways (described above) involving protein kinase G (seeFIG. 8). This suggests that the combination of the two classes of agentswill be more efficacious in relaxing smooth muscle than employing asingle class of agent alone.

[0101] Suitable nitric oxide donors for the practice of the presentinvention include nitroglycerin, sodium nitroprusside, the drug FK 409,FR 144420, 3-morpholinosydnonimine , or linsidomine chlorohydrate,(“SIN-1”); and S-nitroso-N-acetylpenicillamine (“SNAP”). Concentrationsfor these agents are set forth in Table 18. TABLE 18 Therapeutic andPreferred Concentrations of Spasm Inhibitory Agents TherapeuticPreferred Class of Agent Concentrations Concentrations Nitric OxideDonors: (Nanomolar) (Nanomolar) Nitroglycerin 10-10,000 100-1,000 sodiumnitroprusside 10-10,000 100-1,000 SIN-1 10-10,000 100-1,000 SNAP10-10,000 100-1,000 FK 409 (NOR-3) 1-1,000 10-500  FR 144420 (NOR-4)10-10,000 100-5,000

3. Endothelin Receptor Antagonists

[0102] Endothelin is a 21 amino acid peptide that is one of the mostpotent vasoconstrictors known. Three different human endothelinpeptides, designated ET-1, ET-2 and ET-3 have been described whichmediate their physiological effects through at least two receptorsubtypes referred to as ET_(A) and ET_(B) receptors. The heart andvascular smooth muscle contain predominantly ET_(A) receptors and thissubtype is responsible for contraction in these tissues. Furthermore,ET_(A) receptors have often been found to mediate contractile responsesin isolated smooth muscle preparations. Antagonists of ET_(A) receptorshave been found to be potent antagonists of human coronary arterycontractions. Thus, antagonists to the ET_(A) receptor should betherapeutically beneficial in the perioperative inhibition of coronaryvasospasm and may additionally be usefull in inhibition of smooth musclecontraction in urological applications. Miller, R. C., et. al., Trendsin Pharmacol. Sci., Vol. 14, pp. 54-60 (1993).

[0103] Suitable endothelin receptor antagonists include:cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (“BQ 123”);(N,N-hexamethylene)-carbamoyl-Leu-D-Trp-(CHO)-D-Tip-OH (“BQ 610”);(R)2-([R-2-[(s)-2-([1-hexahydro-1H-azepinyl]-carbonyl]amino-4-methyl-pentanoyl)amino-3-(3[1-methyl-1H-indodyl])propionylamino-3(2-pyridyl) propionicacid (“FR 139317”); cyclo(D-Asp-Pro-D-Ile-Leu-D-Trp) (“JKC 301”);cyclo(D-Ser-Pro-D-Val-Leu-D-Trp) (“JK 302”);5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulphonamide(“BMS 182874”); andN-[1-Formyl-N-[N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl]-D-tryptophan(“BQ 610”). Concentrations for a representative three of these agents isset forth in Table 19. TABLE 19 Therapeutic and Preferred Concentrationsof Spasm Inhibitory Agents Therapeutic Preferred Class of AgentConcentrations Concentrations Endothelin Receptor Antagonists:(Nanomolar) (Nanomolar) BQ 123 0.01-1,000  10-1,000 FR 139317  1-100,000 100-10,000 BQ 610 0.01 to 10,000  10-1,000

4. Ca²⁺ Channel Antagonists

[0104] Calcium channel antagonists are a distinct group of drugs thatinterfere with the transmembrane flux of calcium ions required foractivation of cellular responses mediating neuroinflammation. Calciumentry into platelets and white blood cells is a key event mediatingactivation of responses in these cells. Furthermore, the role ofbradykinin receptors and neurokinin receptors (NK₁ and NK₂) in mediatingthe neuroinflammation signal transduction pathway includes increases inintracellular calcium, thus leading to activation of calcium channels onthe plasma membrane. In many tissues, calcium channel antagonists, suchas nifedipine, can reduce the release of arachidonic acid,prostaglandins, and leukotrienes that are evoked by various stimuli.Moncada, S., Flower, R. and Vane, J. in Goodman's and Gilman'sPharmacological Basis of Therapeutics, (7th ed.), MacMillan Publ. Inc.,pp. 660-5 (1995).

[0105] Calcium channel antagonists also interfere with the transmembraneflux of calcium ions required by vascular smooth muscle forcontractions. This effect provides the rationale for the use of calciumchannel antagonists perioperatively during procedures in which the goalis to alleviate vasospasm and promote relaxation of smooth muscle. Thedihydropyridines, including nisoldipine, act as specific inhibitors(antagonists) of the voltage-dependent gating of the L-type subtype ofcalcium channels. Systemic administration of the calcium channelantagonist nifedipine during cardiac surgery previously has beenutilized to prevent or minimize coronary artery vasospasm. Seitelberger,R., et. al., Circulation, Vol. 83, pp. 460-468 (1991).

[0106] Calcium channel antagonists, which are among the anti-spasmagents useful in the present invention, exhibit synergistic effect whencombined with other agents of the present invention. Calcium (Ca²⁺)channel antagonists and nitric oxide (NO) donors interact in achievingvasorelaxation or smooth muscle relaxation, i.e., in inhibiting spasmactivity. A rationale for dual use is based upon the fact that these twoclasses of drugs have different molecular mechanisms of action, may notbe completely effective in achieving relaxation used alone, and may havedifferent time periods of effectiveness. In fact, there are numerousstudies showing that calcium channel antagonists alone cannot achievecomplete relaxation of vascular muscle that has been precontracted witha receptor agonist.

[0107] The effect of nisoldipine, used alone and in combination withnitroglycerin, on spasm of the internal mammary artery (IMA) showed thatthe combination of the two drugs produced a large positive synergisticeffect in the prevention of contraction (Liu et al., 1994). Thesestudies provide a scientific basis for combination of a calcium channelantagonist and nitric oxide (NO) donor for the efficacious prevention ofvasospasm and relaxation of smooth muscle. Examples of systemicadministration of nitroglycerin and nifedipine during cardiac surgery toprevent and treat myocardial ischemia or coronary artery vasospasm havebeen reported (Cohen et al., 1983; Seitelberger et al., 1991).

[0108] Calcium channel antagonists also exhibit synergistic effect withendothelin receptor subtype A (ETA) antagonists. Yanagisawa andcoworkers observed that dihydropyridine antagonists blocked effects ofET-1, an endogenous agonist at the ET_(A) receptor in coronary arterialsmooth muscle, and hence speculated that ET-1 is an endogenous agonistof voltage-sensitive calcium channels. It has been found that thesustained phase of intracellular calcium elevation in smooth musclecells induced by ET_(A) receptor activation requires extracellularcalcium and is at least partially blocked by nicardipine. Thus, theinclusion of a calcium channel antagonist would be expected tosynergistically enhance the actions of an ETA antagonist when combinedin a surgical solution.

[0109] Calcium channel antagonists and ATP-sensitive potassium channelopeners likewise exhibit synergistic action. Potassium channels that areATP-sensitive (K_(ATP)) couple the membrane potential of a cell to thecell's metabolic state via sensitivity to adenosine nucleotides. K_(ATP)channels are inhibited by intracellular ATP but are stimulated byintracellular nucleotide diphosphates. The activity of these channels iscontrolled by the electrochemical driving force to potassium andintracellular signals (e.g., ATP or a G-protein), but are not gated bythe membrane potential per se. K_(ATP) channels hyperpolarize themembrane and thus allow them to control the resting potential of thecell. ATP-sensitive potassium currents have been discovered in skeletalmuscle, brain, and vascular and nonvascular smooth muscle. Bindingstudies with radiolabeled ligands have confirmed the existence ofATP-sensitive potassium channels which are the receptor targets for thepotassium-channel opener drugs such as pinacidil. Opening of thesechannels causes potassium efflux and hyperpolarizes the cell membrane.This hyperpolarization (1) induces a reduction in intracellular freecalcium through inhibition of voltage-dependent Ca²⁺ channels byreducing the probability of opening L-type or T-type calcium channels,(2) restrains agonist induced (at receptor operated channels) Ca²⁺release from intracellular sources through inhibition of inositoltriphosphate (IP₃) formation, and (3) lowers the efficiency of calciumas an activator of contractile proteins. The combined actions of thesetwo classes of drugs (ATP-sensitive potassium channel openers andcalcium channel antagonists) will clamp the target cells into a relaxedstate or one which is more resistant to activation.

[0110] Finally, calcium channel antagonists and tachykinin andbradykinin antagonists exhibit synergistic effects in mediatingneuroinflammation. The role of neurokinin receptors in mediatingneuroinflammation has been established. The neurokinin, (NK₁) andneurokinin₂ (NK₂) receptor (members of the G-protein coupledsuperfamily) signal transduction pathway includes increases inintracellular calcium, thus leading to activation of calcium channels onthe plasma membrane. Similarly, activation of bradykinin₂ (BK₂)receptors is coupled to increases in intracellular calcium. Thus,calcium channel antagonists interfere with a common mechanism involvingelevation of intracellular calcium, part of which enters through L-typechannels. This is the basis for synergistic interaction between calciumchannel antagonists and antagonists to neurokinin and bradykinin₂receptors.

[0111] Suitable calcium channel antagonists for the practice of thepresent invention include nisoldipine, nifedipine, nimodipine,lacidipine, isradipine and amlodipine. Suitable concentrations for theseagents are set forth in Table 20. TABLE 20 Therapeutic and PreferredConcentrations ot Spasm Inhibitory Agents Therapeutic Preferred Class ofAgent Concentrations Concentrations Calcium Channel Antagonists:(Nanomolar) (Nanomolar) nisoldipine 1-10,000 100-1,000 nifedipine1-10,000 100-5,000 nimodipine 1-10,000 100-5,000 lacidipine 1-10,000100-5,000 isradipine 1-10,000 100-5,000 amlodipine 1-10,000 100-5,000

[0112] J. Anti-Restenosis Agents

[0113] Solutions of the present invention utilized for cardiovascularand general vascular procedures may optionally also include ananti-restenosis agent, particularly for angioplasty, rotationalatherectomy and other interventional vascular uses. The following drugsare suitable for inclusion in the previously described cardiovascularand general vascular irrigation solutions when limitation of restenosisis indicated. The following anti-restenosis agents would preferably becombined with anti-spasm, and still more preferably also withanti-painianti-intlammation agents in the solutions of the presentinvention.

1. Antiplatelet Agents

[0114] At sites of arterial injury, platelets adhere to collagen andfibrinogen via specific cell surface receptors, and are then activatedby several independent mediators. A variety of agonists are able toactivate platelets, including collagen, ADP, thromboxane A2, epinephrineand thrombin. Collagen and thrombin serve as primary activators at sitesof vascular injury, while ADP and thromboxane A2 act to recruitadditional platelets into a growing platelet plug. The activatedplatelets degranulate and release other agents which serve aschemoattractants and vasoconstrictors, thus promoting vasospasm andplatelet accumulation. Thus, anti-platelet agents can be antagonistsdrawn from any of the above agonist-receptor targets.

[0115] Since platelets play such an important role in the coagulationcascade, oral antiplatelet agents have been routinely administered topatients undergoing vascular procedures. Indeed, because of thismultiplicity of activators and observations that single antiplateletagents are not effective, some investigators have concluded that acombined treatment protocol is necessary for effectiveness. Recently,Willerson and coworkers reported the intravenous use of 3 combinedagents, ridogrel (an antagonist of thromboxane A2), ketanserin (aserotonin antagonist) and clopidogrel (an ADP antagonist). They foundthat the combination of 3 antagonists inhibited several relevantplatelet functions and reduced neointimal proliferation in a caninecoronary angioplasty model (JACC Abstracts, February 1995). It is stilluncertain which approach to treatment of coronary thrombosis will bemost successful. One possibility would be to include an antiplateletagent and an antithrombotic agent in the cardiovascular and generalvascular solutions of the present invention.

a. Thrombin Inhibitors and Receptor Antagonists

[0116] Thrombin plays a central role in vascular lesion formation and isconsidered the principal mediator of thrombogenesis. Thus, thrombusformation at vascular lesion sites during and after PTCA (percutaneoustransluminal coronary angioplasty) or other vascular procedure iscentral to acute reocclusion and chronic restenosis. This process can beinterrupted by application of direct anti-thrombins, including hirudinand its synthetic peptide analogs, as well as thrombin receptorantagonist peptides (Harker, et al., 1995, Am. J. Cardiol 75, 12B).Thrombin is also a potent growth factor which initiates smooth musclecell proliferation at sites of vascular injury. In addition, thrombinalso plays a role in modulating the effects of other growth factors suchas PDGF (platelet-derived growth factor), and it has been shown thatthrombin inhibitors reduce expression of PDGF mRNA subsequent tovascular injury induced by balloon angioplasty.

[0117] Hirudin is the prototypic direct antithrombin drug since it bindsto the catalytic site and the substrate recognition site (exosite) ofthrombin. Animal studies using baboons have shown that thisproliferative response can be reduced 80% using recombinant hirudin(Ciba-Geigy). Hirulog (Biogen) is a dodecapeptide modeled after hirudin,and binds to the active site of thrombin via a Phe-Pro-Arg linkermolecule. Large clinical trials of hirudin and hirulog are underway totest their efficacy in reducing vascular lesions after PTCA and Phase IIdata on these inhibitors to date is positive, and both drugs arebelieved to be suitable in the solutions of the present invention.Preliminary results of a 1,200 patient trial with repeat angiographicassessment at 6 months to detect restenosis indicated superiorshort-term suppression of ischemic events with hirudin vs. heparin. Anadvantage of this approach is that no significant bleeding complicationswere reported. A sustained-release local hirulog therapy was found todecrease early thrombosis but not neointimal thickening after arterialstenting in pigs. Muller, D. et al., Sustained-Release Local HirulogTherapy Decreases Early Thrombosis but not Neointimal Thickening AfterArterial Stenting, Am. Heart J. 133, No. 2, pp. 211-218, (1996). In thisstudy, hirulog was released from an impregnated polymer placed aroundthe artery.

[0118] Other active anti-thrombin agents being tested which aretheorized to be suitable for the present invention are argatroban (TexasBiotechnology) and efegatran (Lilly). TABLE 21 Therapuetic and PreferredConcentrations of Restenoisis Inhibitory Agents Class of AgentTherapeutic/Preferred Thrombin Inhibitors Concentrations More preferredand Receptor Angtagonists: (Nanomolar) (Nanomolar) hirudin0.00003-3/0.0003-0.3 0.03 hirulog 0.2-20,000/2-2,000 200

b. ADP Receptor Antagonists (Purinoceptor Antagonists)

[0119] Ticlopidine, an analog of ADP, inhibits both thromboxane andADP-induced platelet aggregation. It is likely that ticlopidine blocksinteraction of ADP with its receptor, thereby inhibiting signaltransduction by this G-protein coupled receptor on the surface ofplatelet membranes. A preliminary study showed it to be more effectivethan aspirin in combination with dipyridamole. However, the clinical useof ticlopidine has been limited because it causes neutropenia.Clopidogrel, a ticlopidine analog, is thought to have fewer adverse sideeffects than ticlopidine and is currently being studied for preventionof ischemic events. It is theorized that these agents may be suitablefor use in the solutions of the present invention.

c. Thromboxane Inhibitors and Receptor Antagonists

[0120] Agents currently utilized for conventional methods of treatmentof thrombosis rely upon aspirin, heparin and plasminogen activators.Aspirin irreversibly acetylates cycloxygenase and inhibits the synthesisof thromboxane A2 and prostacyclin. While data support a benefit ofaspirin for PTCA, the underlying efficacy of aspirin is considered asonly partial or modest. This is likely due to platelet activationthrough thromboxane A2 independent pathways that are not blocked byaspirin induced acetylation of cyclooxygenase. Platelet aggregation andthrombosis may occur despite aspirin treatment. Aspirin in combinationwith dipyridarnole has also been shown to reduce the incidence of acutecomplication during PTCA but not the incidence of restenosis. Twothromboxane receptor antagonists appear to be more efficacious thanaspirin and are believed suitable for use in the solutions and methodsof the present invention. Ticlopidine inhibits both thromboxane andADP-induced platelet aggregation. Ridogrel (R68060) is a combinedthromboxane B2 synthetase inhibitor and thromboxane-prostaglandinendoperoxide receptor blocker. It has been compared with salicylatetherapy in an open-pilot study of patients undergoing PTCA administeredin combination with heparin. Timmermans, C., et al., Ridogrel in theSetting of Percutaneous Transluminal Coronary Angioplasty, Am. J.Cardiol. 68, pp. 463-466, (1991). Treatment consisted of administering aslow intravenous injection of 300 mg just prior to the start of the PTCAprocedure and continued orally after 12 hrs with a dose of 300 mg/twicedaily. From this study, ridogrel was found to be primarily successfulsince no early acute reocclusion occurred in 30 patients. Bleedingcomplications did occur in a significant number (34%) of patients, andthis appears to be a complicating factor that would require specialcare. The study confirmed that ridogrel is a potent long-lastinginhibitor of thromboxane B2 synthetase.

2. Inhibitors of Cell Adhesion Molecules a. Selectin Inhibitors

[0121] Selectin inhibitors block the interaction of a selectin with itscognate ligand or receptor. Representative examples of selectin targetsat which these inhibitors would act include, but are not limited to,E-selectin and P-selectin receptors. Upjohn Co. has licensed rights to amonoclonal antibody developed by Cytel Corps that inhibits the activityof P-selectin. The product, CY 1748, is in preclinical development, witha potential indication being restenosis.

b. Integrin inhibitors

[0122] The platelet glycoprotein IIb/IIIa complex is present on thesurface of resting as well as activated platelets. It appears to undergoa transformation during platelet activation which enables it to serve asa binding site for fibrinogen and other adhesive proteins. Mostpromising new antiplatelet agents are directed at this integrin cellsurface receptor which represents a final common pathway for plateletaggregation.

[0123] Several types of agents fit into the class of GPIIb/IIIa integrinantagonists. A monoclonal antibody, c7E3, (CentoRx; Centocor, MalvernPa.) has been intensively studied to date in a 3,000 patient PTCA study.It is a chimeric human/murine hybrid. A 0.25 mg/kg bolus of c7E3followed by 10 μg/min intravenous infuision for 12 hrs produced greaterthan 80% blockade of GPIIb/IIIa receptors for the duration of theinfusion. This was correlated with a greater than 80% inhibition ofplatelet aggregation. The antibody was coadministered with heparin andan increased risk of bleeding was noted. Additional information wasobtained from the EPIC trial which showed a significant reduction in theprimary end point, a composite of death rate, incidence of nonfatalmyocardial infarction and need for coronary revascularization, andsuggested a long term benefit. Tcheng, (1995) Am. Heart J. 130, 673-679.A phase IV study (EPILOG) designed to address safety and efficacy issueswith c7E3 Fab is planned or in progress. This monoclonal antibody canalso be classified as a platelet membrane glycoprotein receptorantagonist directed against the glycoprotein IIb/IIIa receptor.

[0124] The platelet glycoprotein IIb/IIIa receptor blocker, integrelin,is a cyclic heptapeptide that is highly specific for this moleculartarget. In contrast to the antibody, it has a short biologic half-life(about 10 minutes). The safety and efficacy of integrelin was firstevaluated in the Phase II Impact trial. Either 4 or 12 hour intravenousinfusions of 1.0 μg/kg/min of integrelin were utilized (Topol, E., 1995Am. J. Cardiol, 27B-33B). It was provided in combination with otheragents (heparin, aspirin) and was shown to exhibit potent anti-plateletaggregation properties (>80%). A phase III study, the IMPACT II trial,of 4000 patients showed that integrelin markedly reduced ischemic eventsin patients who had undergone Rotablator atherectomy (JACC Abstracts,1996). Suitable concentrations of the drugs c7E3 and integrelin for usein the present invention are set forth below.

[0125] In addition, two peptidomimetics, MK-383 (Merck) and RO 4483(Hoffrnann-LaRoche), have been studied in Phase II clinicals. Sincethese are both small molecules, they have a short half-life and highpotency. However, these seem to also have less specificity, interactingwith other closely related integrins. It is theorized that thesepeptidomimetics may also be suitable for use in the present invention.TABLE 22 Therapuetic and Preferred Concentrations of RestenoisisInhibitory Agents Therapeutic/Preferred Class of Agent ConcentrationsMore preferred Cell Adhesion Inhibitors: (Nanomolar) (Nanomolar) c7E30.5-50,000/5-5,000 500 Integrelin 0.1-10,000/1-1000 x K_(d) 100 x K_(d)

3. Anti-chemotactic Agents

[0126] Anti-chemotactic agents prevent the chemotaxis of inflammatorycells. Representative examples of anti-chemotactic targets at whichthese agents would act include, but are not limited to, F-Met-Leu-Phereceptors, IL-8 receptors, MCP-1 receptors, and MIP-1-α/RANTESreceptors. Drugs within this class of agents are early in thedevelopment stage, but it is theorized that they may be suitable for usein the present invention.

4. Interleukin Receptor Antagonists

[0127] Interleukin receptor antagonists are agents which block theinteraction of an interleukin with its cognate ligand or receptor.Specific receptor antagonists for any of the numerous interleukinreceptors are early in the development process. The exception to this isthe naturally occurring existence of a secreted form of the IL-1receptor, referred to as IL-1 antagonist protein (IL-IAP). Thisantagonist binds IL-1 and has been shown to suppress the biologicalactions of IL-1, and is theorized to be suitable for the practice of thepresent invention.

5. Intracellular Signaling Inhibitors a. Protein Kinase Inhibitors i.Protein Kinase C (PKQ) Inhibitors

[0128] Protein kinase C (PKC) plays a crucial role in cell-surfacesignal transduction for a number of physiological processes. PKCisozymes can be activated as downstream targets resulting from initialactivation of either G-protein coupled receptors (e.g., serotonin,endothelin, etc.) or growth-factor receptors such as PDGF. Both of thesereceptor classes play important roles in mediating vascular spasm andrestenosis subsequent to coronary balloon angioplasty procedures.

[0129] Molecular cloning analysis has revealed that PKC exists as alarge family consisting of at least 8 subspecies (isozymes). Theseisozymes differ substantially in structure and mechanism for linkingreceptor activation to changes in the proliferative response of specificcells. Expression of specific isozymes is found in a wide variety ofcell types, including: platelets, neutrophils, myeloid cells, and smoothmuscle cells. Inhibitors of PKC are therefore likely to effect signalingpathways in several cell types unless the inhibitor shows isozymespecificity. Thus, inhibitors of PKC can be predicted to be effective inblocking the proliferative response of smooth muscle cells and may alsohave an anti-inflammatory effect in blocking neutrophil activation andsubsequent attachment. Several inhibitors have been described andinitial reports indicate an IC₅₀ of 50 nM for calphostin C inhibitoryactivity. G-6203 (also known as Go 6976) is a new, potent PKC inhibitorwith high selectivity for certain PKC isotypes with IC₅₀ values in the2-10 mM range. Concentrations of these and another drug, GF 109203X,also known as Go 6850 or bisindoylmaleimide I (available fromWarner-Lambert), that are believed to be suitable for use in the presentinvention are set forth below. TABLE 23 Therapuetic and PreferredConcentrations of Restenoisis Inhibitory Agents Therapeutic/PreferredClass of Agent Concentrations More preferred Protein Kinase CInhibitors: (Nanomolar) (Nanomolar) calphostin C 0.5-50,000/100-5,000500 GF 109203X 0.1-10,000/1-1,000 100 G-6203 (Go 6976)0.1-10,000/1-1,000 100

ii. Protein Tyrosine Kinase Inhibitors

[0130] Although there is a tremendous diversity among the numerousmembers of the receptors tyrosine-kinase (RTK) family, the signalingmechanisms used by these receptors share many common features.Biochemical and molecular genetic studies have shown that binding of theligand to the extracellular domain of the RTK rapidly activates theintrinsic tyrosine kinase catalytic activity of the intracellular domain(see FIG. 5). The increased activity results in tyrosine-specificphosphorylation of a number of intracellular substrates which contain acommon sequence motif. Consequently, this causes activation of numerous“downstream” signaling molecules and a cascade of intracellular pathwaysthat regulate phospholipid metabolism, arachidonate metabolism, proteinphosphorylation (involving mechanisms other than protein kinases),calcium mobilization and transcriptional activation (see FIG. 2).Growth-factor-dependent tyrosine kinase activity of the RTK cytoplasmicdomain is the primary mechanism for generation of intracellular signalsthat lead to cellular proliferation. Thus, inhibitors have the potentialto block this signaling and thereby prevent the proliferative response(see FIG. 5).

[0131] The platelet-derived growth factor (PDGF) receptor is of greatinterest as a target for inhibition in the cardiovascular field since itis believed to play a significant role both in atherosclerosis andrestenosis. The release of PDGF by platelets at damaged surfaces ofendothelium within blood vessels results in stimulation of PDGFreceptors on vascular smooth muscle cells. As described above, thisinitiates a sequence of intracellular events leading to enhancedproliferation and neointimal thickening. An inhibitor of PDGF kinaseactivity would be expected to prevent proliferation and enhance theprobability of success following cardiovascular and general vascularprocedures. Any of several related tyrphostin compounds have potentialas specific inhibitors of PDGF-receptor tyrosine kinase activity (IC₅₀sin vitro in the 0.5-1.0 μM range), since they have little effect onother protein kinases and other signal transduction systems. To date,only a few of the many tyrphostin compounds are commercially available,and suitable concentrations for these agents as used in the presentinvention are set forth below. In addition, staurosporine has beenreported to demonstrate potent inhibitory effects against severalprotein tyrosine kinases of the src subfamily and a suitableconcentration for this agent as used in the present invention also isset forth below. TABLE 24 Therapuetic and Preferred Concentrations ofRestenoisis Inhibitory Agents Therapeutic/Preferred Class of AgentConcentrations More preferred Protein Kinase Inhibitors: (Nanomolar)(Nanomolar) lavendustin A 10-100,000/100-10,000 10,000 tyrphostin10-100,000/100-20,000 10,000 AG1296 tyrphostin 10-100,000/100-20,00010,000 AG1295 staurosporine 1-100,000/10-10,000  1,000

b. Modulators of Intracellular Protein Tyrosine Phosphatases

[0132] Non-transmembrane protein tyrosine phosphatases (PTPases)containing src-homology₂ SH2 domains are known and nomenclature refersto them as SH-PTP1 and SH-PTP2. In addition, SH-PTP1 is also known asPTP1C, HCP or SHP. SH-PTP2 is also known as PTP1D or PTP2C. Similarly,SH-PTP1 is expressed at high levels in hematopoietic cells of alllineages and all stages of differentiation, and the SH-PTP1 gene hasbeen identified as responsible for the motheaten (me) mouse phenotypeand this provides a basis for predicting the effects of inhibitors thatwould block its interaction with its cellular substrates. Stimulation ofneutrophils with chemotactic peptides is known to result in theactivation of tyrosine kinases that mediate neutrophil responses (Cui,et al., 1994 J. Immunol.) and PTPase activity modulates agonist inducedactivity by reversing the effects of tyrosine kinases activated in theinitial phases of cell stimulation. Agents that could stimulate PTPaseactivity could have potential therapeutic applications asanti-inflammatory mediators.

[0133] These same PTPases have also been shown to modulate the activityof certain RTKs. They appear to counter-balance the effect of activatedreceptor kinases and thus may represent important drug targets. In vitroexperiments show that injection of PTPase blocks insulin stimulatedphosphorylation of tyrosyl residues on endogenous proteins. Thus,activators of PTPase activity could serve to reverse activation ofPDGF-receptor action in restenosis, and are believed to be useful in thesolutions of the present invention. In addition, receptor-linked PTPasesalso function as extracellular ligands, similar to those of celladhesion molecules. The functional consequences of the binding of aligand to the extracellular domain have not yet been defined but it isreasonable to assume that binding would serve to modulate phosphataseactivity within cells (Fashena and Zinn, 1995, Current Biology, 5,1367-1369). Such actions could block adhesion mediated by other cellsurface adhesion molecules (NCAM) and provide an anti-inflammatoryeffect. No drugs have been developed yet for these applications.

c. Inhibitors of SH2 Domains (src Homology₂ Domains).

[0134] SH2 domains, originally identified in the src subfamily ofprotein tyrosine kinases (PTKs), are noncatalytic protein sequences andconsist of about 100 amino acids conserved among a variety of signaltransducing proteins (Cohen, et al., 1995). SH2 domains function asphosphotyrosine-binding modules and thereby mediate criticalprotein-protein associations in signal transduction pathways withincells (Pawson, Nature, 573-580, 1995). In particular, the role of SH2domains has been clearly defined as critical for receptor tyrosinekinase (RTK) mediated signaling such as in the case of theplatelet-derived growth factor (PDGF) receptor.Phosphotyrosine-containing sites on autopho,nhorylated RTKs serve asbinding sites for SH2-proteins and thereby mediate the activation ofbiochemical signaling pathways (see FIG. 2) (Carpenter, G., FASEB J.6:3283-3289, 1992; Sierke, S. and Koland, J. Biochem. 32:10102-10108,1993). The SH2 domains are responsible for coupling the activatedgrowth-factor receptors to cellular responses which include alterationsin gene expression, and ultimately cellular proliferation (see FIG. 5).Thus, inhibitors that will selectively block the effects of activationof specific RTKs expressed on the surface of vascular smooth musclecells are predicted to be effective in blocking proliferation and therestenosis process after PTCA or other vascular procedure. One RTKtarget of current interest is the PDGF receptor.

[0135] At least 20 cytosolic proteins have been identified that containSH2 domains and function in intracellular signaling. The distribution ofSH2 domains is not restricted to a particular protein family, but foundin several classes of proteins, protein kinases, lipid kinases, proteinphosphatases, phospholipases, Ras-controlling proteins and sometranscription factors. Many of the SH2-containing proteins have knownenzymatic activities while others (Grb2 and Crk) function as “linkers”and “adapters” between cell surface receptors and “downstream” effectormolecules (Marengere, L., et al., Nature 369:502-505, 1994). Examples ofproteins containing 1SH2 domains with enzymatic activities that areactivated in signal transduction include, but are not limited to, thesrc subfamily of protein tyrosine kinases (src (pp60^(c-src)), abl, lck,fyn, fgr and others), phospholipaseCγ (PLCγ), phosphatidylinositol3-kinase (PI-3-kinase), p21-ras GTPase activating protein (GAP) and SH2containing protein tyrosine phosphatases (SH-PTPases) (Songyang, et al.,Cell 72, 767-778, 1993). Due to the central role these variousSH2-proteins occupy in transmitting signals from activated cell surfacereceptors into a cascade of additional molecular interactions thatultimately define cellular responses, inhibitors which block specificSH2 protein binding are desirable as agents for a variety of potentialtherapeutic applications.

[0136] In addition, the regulation of many immune/inflammatory responsesis mediated through receptors that transmit signals through non-receptortyrosine kinases containing SH2 domains. T-cell activation via theantigen specific T-cell receptor (TCR) initiates a signal transductioncascade leading to lymphokine secretion and T-cell proliferation. One ofthe earliest biochemical responses following TCR activation is anincrease in tyrosine kinase activity. In particular, neutrophilactivation is in part controlled through responses of the cell surfaceimmunoglobulin G receptors. Activation of these receptors mediatesactivation of unidentified tyrosine kinases which are known to possessSH2 domains. Additional evidence indicates that several src-familykinases (lck, blk, fyn) participate in signal transduction pathwaysleading from cytokine and integrin receptors and hence may serve tointegrate stimuli received from several independent receptor structures.Thus, inhibitors of specific SH2 domains have the potential to blockmany neutrophil functions and serve as anti-inflammatory mediators.

[0137] Efforts to develop drugs targeted to SH2 domains currently arebeing conducted at the biochemical in vitro and cellular level. Shouldsuch efforts be successful, it is theorized that the resulting drugswould be useful in the practice of the present invention.

d. Calcium Channel Antagonists

[0138] Calcium channel antagonists, previously described with relationto spasm inhibitory function, also can be used as anti-restenotic agentsin the cardiovascular and general vascular solutions of the presentinvention. Activation of growth factor receptors, such as PDGF, is knownto result in an increase in intracellular calcium (see FIG. 2). Studiesat the cellular level have shown that actions of calcium channelantagonists are effective at inhibiting mitogenesis of vascular smoothmuscle cells.

6. Synergistic Interactions Derived From Therapeutic Combinations OfAnti-Restenosis Agents And Other Agents Used In Cardiovascular andGeneral Vascular Solutions

[0139] Given the complexity of the disease process associated withrestenosis after PTCA or other cardiovascular or general vasculartherapeutic procedure and the multiplicity of molecular targetsinvolved, blockade or inhibition of a single molecular target isunlikely to provide adequate efficacy in preventing vasospasm andrestenosis (see FIG. 2). Indeed, a number of animal studies targetingdifferent individual molecular receptors and or enzymes have not proveneffective in animal models or have not yielded efficacy for bothpathologies in clinical trials to date. (Freed, M., et al., An IntensivePoly-pharmaceutical Approach to the Prevention of Restenosis: theMevacor, Ace Inhibitor, Colchicine (BIG-MAC) Pilot Trial, J. Am. Coll.of Cardiol. 21, p. 33A, (1993). Serruys, P., et al., PARK: the PostAngioplasty Restenosis Ketanserin Trial, J. Am. Coll. of Cardiol. 21, p.322A, (1993). Therefore, a therapeutic combination of drugs acting ondistinct molecular targets and delivered locally appears necessary forclinical effectiveness in the therapeutic approach to vasospasm andrestenosis. As described below, the rationale for this synergisticmolecular targeted therapy is derived from recent advances inunderstanding fundamental biochemical mechanisms by which vascularsmooth muscle cells in the vessel wall transmit and integrate stimuli towhich they are exposed during PTCA or other vascular interventionalprocedure.

a. “Crosstalk” and Convergence in Major Signaling Pathways

[0140] The molecular switches responsible for cell signaling have beentraditionally divided into two major discrete signaling pathways, eachcomprising a distinct set of protein families that act as transducersfor a particular set of extracellular stimuli and mediating distinctcell responses. One such pathway transduces signals fromneurotransmitters and hormones through G-protein coupled receptors(GPCRs) to produce contractile responses using intracellular targets oftrimeric G proteins and Ca²⁺ (see FIG. 2). These stimuli and theirrespective receptors mediate smooth muscle contraction and may inducevasospasm in the context of PTCA or other cardiovascular or generalvascular therapeutic or diagnostic procedure. Examples of signalingmolecules involved in mediating spasm through the GPCR pathway are 5-HTand endothelin for which antagonists have been included acting via theirrespective G-protein coupled receptors.

[0141] A second major pathway transduces signals from growth factors,such as PDGF, through tyrosine kinases, adaptor proteins and the Rasprotein into regulation of cell proliferation and differentiation (seeFIGS. 2 and 5). This pathway may also be activated during PTCA or othercardiovascular or general vascular procedure leading to a high incidenceof vascular smooth muscle cell proliferation. An example of a restenosisdrug target is the PDGF-receptor.

[0142] Signals transmitted from neurotransmitters and hormones stimulateeither of two classes of receptors: G-protein-coupled receptors,composed of seven-helix transmembrane regions, or ligand-gated ionchannels. “Downstream” signals from both kinds of receptors converge oncontrolling the concentration of cytoplasmic Ca²⁺ which triggerscontraction in smooth muscle cells (see FIG. 2). Each GPCR transmembranereceptor activates a specific class of trimeric G proteins, includingG_(q), G_(i) or many others. G_(α) and/or G_(βγ) subunits activatephospholipase C_(β), resulting in activation of protein kinase C (PKC)and an increase in the levels of cytoplasmic calcium by release ofcalcium from intracellular stores.

[0143] Growth factor signaling, such as mediated by PDGF, converges onregulation of cell growth. This pathway depends upon phosphorylation oftyrosine residues in receptor tyrosine kinases and “downstream” enzymes(phospholipase C_(γ), discussed above with regard to tyrosine kinases).Activation of the PDGF-receptor also leads to stimulation of PKC andelevation of intracellular calcium, common steps shared by the GPCRs(see FIG. 2). It is now recognized that ligand-independent “crosstalk”can transactivate tyrosine kinase receptor pathways in response tostimulation of GPCRS. Recent work has identified Shc, an adaptor proteinin the tyrosine kinase/Ras pathway, as a key intermediary protein thatrelays messages from the GPCR pathway described above to the tyrosinekinase pathway (see FIG. 2) (Lev et al., 1995, Nature 376:737).Activation of Shc is calcium dependent. Thus, a combination of selectiveinhibitors which blocks transactivation of a common signaling pathwayleading to vascular smooth muscle cell proliferation will actsynergistically to prevent spasm and restenosis after PTCA or othercardiovascular or general vascular procedure. Specific examples arebriefly detailed below.

b. Synergistic Interactions between PKC Inhibitors and Calcium ChannelAntagonists

[0144] In this case synergistic interactions among PKC inhibitors andcalcium channel antagonists in achieving vasorelaxation and inhibitionof proliferation occur due to “crosstalk” between GPCR and tyrosinekinase signaling pathways (see FIG. 2). A rationale for dual use isbased upon the fact that these drugs have different molecular mechanismsof action. As described above, GPCR stimulation results in activation ofprotein kinase C and an increase in the levels of cytoplasmic calcium byrelease of calcium from intracellular stores. Calcium-activated PKC is acentral control point in the transmission of extracellular responses.“Crosstalk” from GPCR stimulated pathways through PKC can lead tomitogenesis of vascular smooth muscle cells and thus calcium channelantagonists will have the dual action of directly blocking spasm andfurther preventing activation of proliferation by inhibiting Shcactivation. Conversely, the PKC inhibitor acts on part of the pathwayleading to contraction.

c. Synergistic Effects of PKC Inhibitors, 5-HT₂ Antagonists and ET_(A)Antagonists

[0145] The 5-HT₂ receptor family contains three members designated5-HT_(2A), 5-HT_(2B), and 5-HT_(2C), all of which share the commonproperty of being coupled to phosphotidylinositol turnover and increasesin intracellular calcium (Hoyer et al., 1988, Hartig et al., 1989). Thedistribution of these receptors includes vascular smooth muscle andplatelets and, due to their localization, these 5-HT receptors areimportant in mediating spasm, thrombosis and restenosis. It has beenfound that the sustained phase of intracellular calcium elevation insmooth muscle cells induced by ET_(A) receptor activation requiresextracellular calcium and is at least partially blocked by nicardipine.Since activation of both 5-HT₂ receptors and ET_(A) receptors ismediated through calcium, the inclusion of a PKC inhibitor is expectedto synergistically enhance the actions of antagonists to both of thesereceptors when combined in a surgical solution (see FIGS. 2 and 4).

d. Synergistic Effects of Protein Tyrosine Kinase Inhibitors and CalciumChannel Antagonists

[0146] The mitogenic effect of PDGF (or basic fibroblast growth factoror insulin-like-growth-factor-1) is mediated through receptors thatpossess intrinsic protein tyrosine kinase activity. The substrates forPDGF phosphorylation are many and lead to activation ofmitogen-activated protein kinases (MAPK) and ultimately proliferation(see FIG. 5). The endothelin, 5-HT and thrombin receptors, which aremembers of the G-protein coupled superfamily, trigger a signaltransduction pathway which includes increases in intracellular calcium,leading to activation of calcium channels on the plasma membrane. Thus,calcium channel antagonists interfere with a common mechanism employedby these GPCRs. It has recently been shown that activation of certainGPCRs, including endothelin and bradykinin, leads to a rapid increase intyrosine phosphorylation of a number of intracellular proteins. Some ofthe proteins phosphorylated parallel those known necessary for mitogenicstimulation. The rapidity of the process was such that changes weredetectable in seconds and the targets acted upon likely play a role inmitogenesis. These tyrosine phosphorylation events were not blocked by aselective PKC inhibitor or apparently mediated by increasedintracellular calcium. Thus, since two independent pathways, the GPCRand tyrosine phosphorylation pathways, can drive the vascular smoothmuscle cells into a proliferative state, it is necessary to block bothindependent signaling arms. This is the basis for the synergisticinteraction between -calcium channel antagonists and tyrosine kinaseinhibitors in the surgical solution. Because the actions of the proteintyrosine kinase inhibitors in preventing vascular smooth muscle cellproliferation occur via independent molecular pathways (described above)from those involving calcium and protein kinase C, the combination ofthe two classes of drugs, calcium channel antagonists and proteintyrosine kinase inhibitors, is expected to be more efficacious ininhibiting spasm and restenosis than employing either single class ofdrug alone.

e. Synergistic Effects of Protein Tyrosine Kinase Inhibitors andThrombin Receptor Antagonists

[0147] Thrombin mediates its action via the thrombin receptor, anothermember of the GPCR superfamily. Binding to the receptor stimulatesplatelet aggregation, smooth muscle cell contraction and mitogenesis.Signal transduction occurs through multiple pathways: activation ofphospholipse (PLC) through G proteins and activation of tyrosinekinases. The activation of tyrosine kinase activity is also essentialfor mitogenesis of the vascular smooth muscle cells. Experiments haveshown that inhibition with a specific tyrosine kinase inhibitor waseffective in blocking thrombin-induced mitosis, although there were noeffects on the PLC pathway as monitored by measurement of intracellularcalcium (Weiss and Nucitelli, 1992, J. Biol. Chem. 267:5608-5613).Because the actions of the protein tyrosine kinase inhibitors inpreventing vascular smooth muscle cell proliferation occur viaindependent molecular pathways (described above) from those involvingcalcium and protein kinase C, the combination of protein tyrosine kinaseinhibitors and thrombin receptor antagonists is anticipated to be moreefficacious in inhibiting platelet aggregation, spasm and restenosisthan employing either class of agent alone.

VI. METHOD OF APPLICATION

[0148] The solution of the present invention has applications for avariety of operative/interventional procedures, including surgical,diagnostic and therapeutic techniques. The irrigation solution isperioperatively applied during arthroscopic surgery of anatomic joints,urological procedures, cardiovascular and general vascular diagnosticand therapeutic procedures and for general surgery. As used herein, theterm “perioperative” encompasses application intraprocedurally, pre- andintraprocedurally, intra- and postprocedurally, and pre-, intra- andpostprocedurally. Preferably the solution is applied preprocedurallyand/or postprocedurally as well as intraprocedurally. Such proceduresconventionally utilize physiologic irrigation fluids, such as normalsaline or lactated Ringer's, applied to the surgical site by techniqueswell known to those of ordinary skill in the art. The method of thepresent invention involves substituting theanti-pain/anti-inflammatory/anti-spasm/anti-restenosis irrigationsolutions of the present invention for conventionally applied irrigationfluids. The irrigation solution is applied to the wound or surgical siteprior to the initiation of the procedure, preferably before tissuetrauma, and continuously throughout the duration of the procedure, topreemptively block pain and inflammation, spasm and restenosis. As usedherein throughout, the term “irrigation” is intended to mean theflushing of a wound or anatomic structure with a stream of liquid. Theterm “application” is intended to encompass irrigation and other methodsof locally introducing the solution of the present invention, such asintroducing a gellable version of the solution to the operative site,with the gelled solution then remaining at the site throughout theprocedure. As used herein throughout, the term “continuously” isintended to also include situations in which there is repeated andfrequent irrigation of wounds at a frequency sufficient to maintain apredetermined therapeutic local concentration of the applied agents, andapplications in which there may be intermittent cessation of irrigationfluid flow necessitated by operating technique.

[0149] The concentrations listed for each of the agents within thesolutions of the present invention are the concentrations of the agentsdelivered locally, in the absence of metabolic transformation, to theoperative site in order to achieve a predetermined level of effect atthe operative site. It is understood that the drug concentrations in agiven solution may need to be adjusted to account for local dilutionupon delivery. For example, in the cardiovascular application, if oneassumes an average human coronary artery blood flow rate of 80 cc perminute and an average delivery rate for the solution of 5 cc per minutevia a local delivery catheter (i.e., a blood flow-to-solution deliveryratio of 16 to 1), one would require that the drug concentrations withinthe solution be increased 16-fold over the desired in vivo drugconcentrations. Solution concentrations are not adjusted to account formetabolic transformations or dilution by total body distribution becausethese circumstances are avoided by local delivery, as opposed to oral,intravenous, subcutaneous or intramuscular application.

[0150] Arthroscopic techniques for which the present solution may beemployed include, by way of non-limiting example, partial meniscectomiesand ligament reconstructions in the knee, shoulder acromioplasties,rotator cuff debridements, elbow synovectomies, and wrist and anklearthroscopies. The irrigation solution is continuously suppliedintraoperatively to the joint at a flow rate sufficient to distend thejoint capsule, to remove operative debris, and to enable unobstructedintra- articular visualization.

[0151] A suitable irrigation solution for control of pain and edemaduring such arthroscopic techniques is provided in Example I hereinbelow. For arthroscopy, it is preferred that the solution include acombination, and preferably all, or any of the following: a serotonin2receptor antagonist, a serotonin₃ receptor antagonist, a histamine₁receptor antagonist, a serotonin receptor agonist acting on the 1A, 1B,1D, 1F and/or 1E receptors, a bradykinin, receptor antagonist, abradykinin₂ receptor antagonist, and a cyclooxygenase inhibitor.

[0152] This solution utilizes extremely low doses of these pain andinflammation inhibitors, due to the local application of the agentsdirectly to the operative site during the procedure. For example, lessthan 0.05 mg of amitriptyline (a suitable serotonin₂ and histamine₁“dual” receptor antagonist) are needed per liter of irrigation fluid toprovide the desired effective local tissue concentrations that wouldinhibit 5-HT₂ and H₁ receptors. This dosage is extremely low relative tothe 10-25 mg of oral amitriptyline that is the usual starting dose forthis drug. This same rationale applies to the anti-spasm andanti-restenosis agents which are utilized in the solution of the presentinvention to reduce spasm associated with urologic, cardiovascular andgeneral vascular procedures and to inhibit restenosis associated withcardiovascular and general vascular procedures. For example, less than0.2 mg of nisoldipine (a suitable calcium channel antagonist) isrequired per liter of irrigation fluid to provide the desired effectivelocal tissue concentrations that would inhibit the voltage-dependentgating of the L-subtype of calcium channels. This dose is extremely lowcompared to the single oral dose of nisoldipine which is 20 to 40 mg.

[0153] In each of the surgical solutions of the present invention, theagents are included in low concentrations and are delivered locally inlow doses relative to concentrations and doses required withconventional methods of drug administration to achieve the desiredtherapeutic effect. It is impossible to obtain an equivalent therapeuticeffect by delivering similarly dosed agents via other (i.e.,intravenous, subcutaneous, intramuscular or oral) routes of drugadministration since drugs given systemically are subject to first- andsecond-pass metabolism.

[0154] For example, using a rat model of arthroscopy, the inventorsexamined the ability of amitriptyline, a 5-HT₂ antagonist, to inhibit5-HT-induced plasma extravasation in the rat knee in accordance with thepresent invention. This study, described more fully below in ExampleXII. compared the therapeutic dosing of amitriptyline delivered locally(i.e., intra-articularly) at the knee and intravenously. The resultsdemonstrated that intra-articular administration of amitriptylinerequired total dosing levels approximately 200-fold less than wererequired via the intravenous route to obtain the same therapeuticeffect. Given that only a small fraction of the drug deliveredintra-articularly is absorbed by the local synovial tissue, thedifference in plasma drug levels between the two routes ofadministration is much greater than the difference in totalamitriptyline dosing levels.

[0155] Practice of the present invention should be distinguished fromconventional intra-articular injections of opiates and/or localanesthetics at the completion of arthroscopic or “open” joint (e.g.,knee, shoulder, etc.) procedures. The solution of the present inventionis used for continuous infusion throughout the surgical procedure toprovide preemptive inhibition of pain and inflammation. In contrast, thehigh concentrations necessary to achieve therapeutic efficacy with aconstant infusion of local anesthetics, such as lidocaine (0.5-2%solutions), would result in profound systemic toxicity.

[0156] Upon completion of the procedure of the present invention, it maybe desirable to inject or otherwise apply a higher concentration of thesame pain and inflammation inhibitors as used in the irrigation solutionat the operative site, as an alternative or supplement to opiates.

[0157] The solution of the present invention also has application incardiovascular and general vascular diagnostic and therapeuticprocedures to potentially decrease vessel wall spasm, plateletaggregation, vascular smooth muscle cell proliferation and nociceptoractivation produced by vessel manipulation. Reference herein to arterialtreatment is intended to encompass the treatment of venous graftsharvested and placed in the arterial system. A suitable solution forsuch techniques is disclosed in Example II herein below. Thecardiovascular and general vascular solution preferably includes anycombination, and preferably all, of the following: a 5-HT₂ receptorantagonist (Saxena, P. R., et. al., Cardiovascular Effects of SerotoninInhibitory Agonists and Antagonists, J Cardiovasc Pharmacol 15 (Suppl.7), pp. S17- S34 (1990); Douglas, 1985); a 5-HT₃ receptor antagonist toblock activation of these receptors on sympathetic neurons and C-fibernociceptive neurons in the vessel walls, which has been shown to producebrady- and tachycardia (Saxena et. al. 1990); a bradykinin₁ receptorantagonist; and a cyclooxygenase inhibitor to prevent production ofprostaglandins at tissue injury sites and thereby decreasing pain andinflammation. In addition, the cardiovascular and general vascularsolution also preferably will contain a serotonin_(1B) (also known asserotonin_(1D) _(β) ) antagonist because serotonin has been shown toproduce significant vascular spasm via activation of the serotoniniBreceptors in humans. Kaurnarn, A. J., et al., Variable Participation of5-HT1-Like Receptors and 5-HT2 Receptors in Serotonin-InducedContraction of Human Isolated Coronary Arteries, Circulation 90, pp.1141-53 (1994). This excitatory action of serotoninIB receptors invessel walls, resulting in vasoconstriction, is in contrast to thepreviously-discussed inhibitory action of serotonin_(1B) receptors inneurons. The cardiovascular and general vascular solution of the presentinvention also may suitably include one or more of the anti-restenosisagents disclosed herein that reduce the incidence and severity ofpost-procedural restenosis resulting from, -for example, angioplasty orrotational atherectomy.

[0158] The solution of the present invention also has utility forreducing pain and inflammation associated with urologic procedures, suchas trans-urethral prostate resection and similar urologic procedures.References herein to application of solution to the urinary tract or tothe urological structures is intended to include application to theurinary tract per se, bladder and prostate and associated structures.Studies have demonstrated that serotonin, histamine and bradykininproduce inflammation in lower urinary tract tissues. Schwartz, M. M.,et. al., Vascular Leakage in the Kidney and Lower Urinary Tract: Effectsof Histamine, Serotonin and Bradykinin, Proc Soc Exp Biol Med 140, pp.535-539 (1972). A suitable irrigation solution for urologic proceduresis disclosed in Example III herein below. The solution preferablyincludes a combination, and preferably all, of the following: ahistamine₁ receptor antagonist to inhibit histamine-induced pain andinflammation; a 5-HT₃ receptor antagonist to block activation of thesereceptors on peripheral C-fiber nociceptive neurons; a bradykinin,antagonist; a bradykinin₂ antagonist; and a cyclooxygenase inhibitor todecrease pain/inflammation produced by prostaglandins at the tissueinjury sites. Preferably an anti-spasm agent is also included to preventspasm in the urethral canal and bladder wall.

[0159] Some of the solutions of the present invention may suitably alsoinclude a gelling agent to produce a dilute gel. This gellable solutionmay be applied, for example, within the urinary tract or an arterialvessel to deliver a continuous, dilute local predetermined concentrationof agents.

[0160] The solution of the present invention may also be employedperioperatively for the inhibition of pain and inflammation in surgicalwounds, as well as to reduce pain and inflammation associated withburns. Bums result in the release of a significant quantity of biogenicamines, which not only produce pain and inflammation, but also result inprofound plasma extravasation (fluid loss), often a life-threateningcomponent of severe burns. Holliman, C. J., et. al., The Effect ofKetanserin, a Specific Serotonin Antagonist, on Burn Shock HemodynamicParameters in a Porcine Burn Model, J Trauma 23, pp. 867-871 (1983). Thesolution disclosed in Example I for arthroscopy may also be suitablyapplied to a wound or burn for pain and inflammation control, and forsurgical procedures such as arthroscopy. The agents of the solution ofExample I may alternately be included in a paste or salve base, forapplication to the burn or wound.

VII. EXAMPLES

[0161] The following are several formulations in accordance with thepresent invention suitable for certain operative procedures followed bya summary of three clinical studies utilizing the agents of the presentinvention.

A. Example I Irrigation Solution for Arthroscopy

[0162] The following composition is suitable for use in anatomic jointirrigation during arthroscopic procedures. Each drug is solubilized in acarrier fluid containing physiologic electrolytes, such as normal salineor lactated Ringer's solution, as are the remaining solutions describedin subsequent examples. Concentration Class of (Nanomolar): Most AgentDrug Therapeutic Preferred Preferred serotonin₂ amitriptyline  0.1-1,000 50-500 100 antagonist serotonin₃ meto-    10-10,000   200-2,000 1,000  antagonist clopramide histamine₁ amitriptyline  0.1-1,000  50-500 200antagonist sero- sumatriptan    1-1,000  10-200  50 tonin_(1A,1B,1D,1F)agonist bradykinin₁ [des-Arg¹⁰]    1-1,000  50-500 200 antagonistderivative of HOE 140 bradykinin₂ HOE 140    1-1,000  50-500 200antagonist

B. Example II Irrigation Solution for Cardiovascular and GeneralVascular Therapeutic and Diagnostic Procedures

[0163] The following drugs and concentration ranges in solution in aphysiologic carrier fluid are suitable for use in irrigating operativesites during cardiovascular and general vascular procedures.Concentration Class of (Nanomolar): Most Agent Drug TherapeuticPreferred Preferred serotonin₂ trazodone  0.1-2,000  50-500 200antagonist serotonin₃ meto-    10-10,000   200-2,000 1,000   antagonistclopramide serotonin_(1B) yohimbine  0.1-1,000  50-500 200 antagonistbradykinin₁ [des-Arg¹⁰]    1-1,000  50-500 200 antagonist derivative ofHOE 140 Cyclooxy- ketorolac   100-10,000   500-5,000 3,000   genaseinhibitor

C. Example III Irrigation Solution for Urologic Procedures

[0164] The following drugs and concentration ranges in solution in aphysiologic carrier fluid are suitable for use in irrigating operativesites during urologic procedures. Concentration Class of (Nanomolar):Most Agent Drug Therapeutic Preferred Preferred histamine₁ terfenadine 0.1-1,000  50-500 200 antagonist serotonin₃ meto-    10-10,000  200-2,000 1,000   antagonist clopramide bradykinin₁ [des-Arg¹⁰]   1-1,000  50-500 200 antagonist derivative of HOE 140 bradykinin₂ HOE140    1-1,000  50-500 200 antagonist cyclooxy-   100-10,000   500-5,0003,000   genase inhibitor

D. Example IV Irrigation Solution for Arthroscopy, Burns, GeneralSurgical Wounds and Oral/Dental Applications

[0165] The following composition is preferred for use in anatomicirrigation during arthroscopic and oral/dental procedures and themanagement of burns and general surgical wounds. While the solution setforth in Example I is suitable for use with the present invention, thefollowing solution is even more preferred because of expected higherefficacy. Concentration Most Class of (Nanomolar): Pre- Agent DrugTherapeutic Preferred ferred serotonin₂ Amitrip-  0.1-1,000  50-500  200 antagonist tyline serotonin₃ metoclop-    10-10,000   200-2,000 1,000 antagonist ramide histamine₁ amitrip-  0.1-1,000  50-500   200antagonist tyline seroton- sumat-    1-1,000  10-200   100in_(1A-,1B,1D,1F) riptan agonist cyclooxy- ketorolac   100-10,000  500-5,000  3,000 genase inhibitor neurokinin₁ GR 82334    1-1,000 10-500   200 antagonist neurokinin₂ (±) SR    1-1,000  10-500   200antagonist 48968 purine_(2X) PPADS    100-100,000  10,000-100,000 50,000antagonist ATP- (−)    1-10,000   100-1,000   500 sensitive pinacidil K⁺channel agonist Ca²⁺channel nifedipine    1-10,000   100-5000  1,000antagonist kallikrein aprotinin  0.1-1,000  50-500   200 inhibitor

E. Example V Alternate Irrigation Solution for Cardiovascular andGeneral Vascular Therapeutic and Diagnostic Procedures

[0166] The following drugs and concentration ranges in solution in aphysiologic carrier fluid are preferred for use in irrigating operativesites during cardiovascular and general vascular procedures. Again, thissolution is preferred relative to the solution set forth in Example IIabove for higher efficacy. Concentration Most Class of (Nanomolar): Pre-Agent Drug Therapeutic Preferred ferred serotonin₂ trazodone  0.1-2,000 50-500   200 antagonist cyclooxy- ketorolac   100-10,000   500-5,0003,000 genase inhibitor endothelin BQ 123  0.01-1,000   10-1,000   500antagonist ATP- (−)    1-10,000   100-1,000   500 sensitive pinacidil K⁺channel agonist Ca²⁺channel nisold-    1-10,000   100-1,000   500antagonist ipine nitric oxide SIN-1    10-10,000   100-1,000   500 donor

F. Example VI Alternate Irrigation Solution for Urologic Procedures

[0167] The following drugs and concentration ranges in solution in aphysiologic carrier fluid are preferred for use in irrigating operativesites during urologic procedures. The solution is believed to have evenhigher efficacy than the solution set forth in prior Example III.Concentration Most Class of (Nanomolar): Pre- Agent Drug TherapeuticPreferred ferred serotonin₂ LY 53857  0.1-500  1-100    50 antagonisthistamine₁ terfen-  0.1-1,000  50-500   200 antagonist adine cyclooxy-ketorolac   100-10,000   500-5,000  3,000 genase inhibitor neurokinin₁SR 48968    1-1,000  10-500   200 antagonist purine_(2X) PPADS   100-100,000  10,000-100,000 50,000 antagonist ATP- (−)    1-10,000  100-1,000   500 sensitive pinacidil K⁺ channel agonist Ca²⁺channelnifedipine    1-10,000   100-5000  1,000 antagonist kallikrein aprotinin 0.1-1,000  50-500   200 inhibitor nitric oxide SIN-1    10-10,000  100-1,000   500 donor

G. Example VII Cardiovascular and General Vascular Anti-RestenosisIrrigation Solution

[0168] The following drugs and concentration ranges in solution in aphysiologic carrier fluid are preferred for use in irrigation duringcardiovascular and general vascular therapeutic and diagnosticprocedures. The drugs in this preferred solution may also be added atthe same concentration to the cardiovascular and general vascularirrigation solutions of Examples II and V described above or ExampleVIII described below for preferred anti-spasmodic, anti-restenosis,anti-pain/anti-inflammation solutions. Concentration (Nanomolar): MostClass of Agent Drug Therapeutic Preferred Preferred thrombin inhibitorhirulog 0.2-20,000 2-2,000 200 glycoprotein IIb/IIIa integrelin0.1-10,000 × Kd 1-1000 × Kd 100 × Kd receptor blocker PKC inhibitor GF109203X* 0.1-10,000 1-1,000 200 protein tyrosine tyrphostin 10-100,000100-20,000 10,000 kinase inhibitor AG1296

H. Example VIII Alternate Irrigation Solution for Cardiovascular andGeneral Vascular Therapeutic and Diagnostic Procedures

[0169] An additional preferred solution for use in cardiovascular andgeneral vascular therapeutic and diagnostic procedures is formulated thesame as the previously described formulation of Example V, except thatthe nitric oxide (NO donor) SIN-1 is replaced by a combination of twoagents, FK 409 (NOR-3) and FR 144420 (NOR-4), at the concentrations setforth below: Concentration Class of (Nanomolar): Most Agent DrugTherapeutic Preferred Preferred NO donor FK 409 (NOR-3) 1-1,000 10-500250 NO donor FR 144420 10-10,000 100-5,000 1,000 (NOR-4)

I. Example IX Alternate Irrigation Solution for Arthroscopy, GeneralSurgical Wounds, Burns and Oral/Dental Applications

[0170] An alternate preferred solution for use in irrigation ofarthroscopic, general surgical and oral/dental applications isformulated the same as in the previously described Example IV, with thefollowing substitution, deletion and additions at the concentrations setforth below:

[0171] 1) amitriptyline is replaced by mepyramine as the HI antagonist;

[0172] 2) the kallikrein inhibitor, aprotinin, is deleted;

[0173] 3) a bradykinin₁ antagonist, [leu⁹] [des-Arg¹⁰] kalliden, isadded;

[0174] 4) a bradykinin₂ antagonist, HOE 140, is added; and

[0175] 5) a μ-opioid agonist, fentanyl, is added. Concentration Class of(Nanomolar): Most Agent Drug Therapeutic Preferred Preferred H₁antagonist mepyramine 0.1-1,000  5-200 100 bradykinin₁ [leu⁹][des-Arg¹⁰]0.1-500 10-200 100 antagonist kalliden bradykinin₂ HOE 140 1-1,00050-500 200 antagonist μ-opioid fentanyl 0.1-500 10-200 100 agonist

J. Example X Alternate Irrigation solution for Urologic Procedures

[0176] An alternate preferred solution for use in irrigation duringurologic procedures is formulated the same as in the previouslydescribed Example VI with the following substitution, deletion andadditions at the concentrations set forth below:

[0177] 1) SIN-1 is replaced as the NO donor by a combination of twoagents:

[0178] a) FK 409 (NOR-3); and

[0179] b) FR 144420 (NOR-4);

[0180] 2) the kallikrein inhibitor, aprotinin, is deleted;

[0181] 3) a bradykininI antagonist, [leu⁹] [des-Arg¹⁰] kalliden, isadded; and

[0182] 4) a bradykinin₂ antagonist, HOE 140, is added. ConcentrationClass of (Nanomolar): Most Agent Drug Therapeutic Preferred Preferred NOdonor FK 409 (NOR-3) 1-1,000 10-500 250 NO donor FR 144420 10-10,000100-5,000 1,000 (NOR-4) bradykinin₁ [leu⁹][des-Arg¹⁰] 0.1-500 10-200 100antagonist kalliden bradykinin₂ HOE 140 1-1,000 50-500 200 antagonist

K. Example XI Balloon Dilatation of Normal Iliac Arteries in the NewZealand White Rabbit and the Influence of Histamine/Serotonin ReceptorBlockade on the Response

[0183] The purpose of this study was twofold. First, a new in vivo modelfor the study of arterial tone was employed. The time course of arterialdimension changes before and after balloon angioplasty is describedbelow. Second, the role of lustamine and serotonin together in thecontrol of arterial tone in this setting was then studied by theselective infusion of histamine and serotonin receptor blocking agentsinto arteries before and after the angioplasty injury.

1. Design Considerations

[0184] This study was intended to describe the time course of change inarterial lumen dimensions in one group of arteries and to evaluate theeffect of histamine/serotonin receptor blockade on these changes in asecond group of similar arteries. To facilitate the comparison of thetwo different groups, both groups were treated in an identical mannerwith the exception of the contents of an infusion performed during theexperiment. In control animals (arteries), the infusion was normalsaline (the vehicle for test solution). The histamine/serotonin receptorblockade treated arteries received saline containing the receptorantagonists at the same rate and at the same part of the protocol ascontrol animals. Specifically, the test solution included: (a) theserotonin₃ antagonist metoclopramide at a concentration of 16.0 μM; (b)the serotonin₂ antagonist trazodone at a concentration of 1.6 μM; and(c) the histamine antagonist promethazine at concentrations of 1.0 μM,all in normal saline. Drug concentrations within the test solution were16-fold greater than the drug concentrations delivered at the operativesite due to a 16 to 1 flow rate ratio between the iliac artery (80 ccper minute) and the solution delivery catheter (5 cc per minute). Thisstudy was performed in a prospective, randomized and blinded manner.Assignment to the specific groups was random and investigators wereblinded to infusion solution contents (saline alone or saline containingthe histamine/serotonin receptor antagonists) until the completion ofthe angiographic analysis.

2. Animal Protocol

[0185] This protocol was approved by the Seattle Veteran Affairs MedicalCenter Committee on Animal Use and the facility is fully accredited bythe American Association for Accreditation of Laboratory Animal Care.The iliac arteries of 3-4 kg male New Zealand white rabbits fed aregular rabbit chow were studied. The animals were sedated usingintravenous xylazine (5 mg/kg) and ketamine (35 mg/kg) dosed to effectand a cutdown was performed in the ventral midline of the neck toisolate a carotid artery. The artery was ligated distally, anarteriotomy performed and a 5 French sheath was introduced into thedescending aorta. Baseline blood pressure and heart rate were recordedand then an angiogram of the distal aorta and bilateral iliac arterieswas recorded on 35 mm cine film (frame rate 15 per second) using handinjection of iopamidol 76% (Squibb Diagnostics, Princeton, N.J.) intothe descending aorta. For each angiogram, a calibration object wasplaced in the radiographic field of view to allow for correction formagnification when diameter measurements were made. A 2.5 Frenchinfusion catheter (Advanced Cardiovascular Systems, Santa Clara, Calif.)was placed through the carotid sheath and positioned 1-2 cm above theaortic bifurcation. Infusion of the test solution—either saline alone orsaline containing the histamine/serotonin receptor antagonists—wasstarted at a rate of 5 cc per minute and continued for 15 minutes. At 5minutes into the infusion, a second angiogram was performed using thepreviously described technique then a 2.5 mm balloon angioplastycatheter (the Lightning, Cordis Corp., Miami, Fla.) was rapidly advancedunder fluoroscopic guidance into the left and then the right iliacarteries. In each iliac the balloon catheter was carefully positionedbetween the proximal and distal deep femoral branches using bonylandmarks and the balloon was inflated for 30 seconds to 12 ATM ofpressure. The balloon catheter was inflated using a dilute solution ofthe radiographic contrast agent so that the inflated balloon diametercould be recorded on cine film. The angioplasty catheter was rapidlyremoved and another angiogram was recorded on cine film at a mean of 8minutes after the infusion was begun. The infusion was continued untilthe 15 minute time point and another angiogram (the fourth) wasperformed. Then the infusion was stopped (a total of 75 cc of solutionhad been infused) and the infusion catheter was removed. At the 30minute time point (15 minutes after the infusion was stopped), a finalangiogram was recorded as before. Blood pressure and heart rate wererecorded at the 15 and 30 minute time points immediately before theangiograms. After the final angiogram, the animal was euthanized with anoverdose of the anesthetic agents administered intravenously and theiliac arteries were retrieved and immersion fixed in formation forhistologic analysis.

3. Angiographic Analysis

[0186] The angiograms were recorded on 35 mm cine film at a frame rateof 15 per second. For analysis, the angiograms were projected from aVanguard projector at a distance of 5.5 feet. Iliac artery diameters atprespecified locations relative to the balloon angioplasty site wererecorded based on hand held caliper measurement after correction formagnification by measurement of the calibration object. Measurementswere made at baseline (before test solution infusion was begun), 5minutes into the infusion, immediately post balloon angioplasty (a meanof 8 minutes after the test solution was begun), at 15 minutes Oustbefore the infusion was stopped) and at 30 minutes (15 minutes after theinfusion was stopped). Diameter measurements were made at three sites ineach iliac artery: proximal to the site of balloon dilatation, at thesite of balloon dilatation and just distal to the site of balloondilatation.

[0187] The diameter measurements were then converted to areameasurements by the formula:

Area=(Pi)(Diameter)/4.

[0188] For calculation of vasoconstriction, baseline values were used torepresent the maximum area of the artery and percent vasoconstrictionwas calculated as: % Vasoconstriction={(Baseline area−Later time pointarea)/Baseline area}×100.

4. Statistical Methods

[0189] All values are expressed as mean±1 standard error of the mean.The time course of vasomotor response in control arteries was assessedusing one way analysis of variance with correction for repeatedmeasures. Post hoc comparison of data between specific time points wasperformed using the Scheffe test. Once the time points at whichsignificant vasoconstriction occurred had been determined in controlarteries, the control and histamine/serotonin receptor antagonisttreated arteries were compared at those time points where significantvasoconstriction occurred in control arteries using multiple analysis ofvariance with treatment group identified as an independent variable. Tocompensate for the absence of a single apriori stated hypothesis, a pvalue <0.01 was considered significant. Statistics were performed usingStatistica for Windows, version 4.5, (Statsoft, Tulsa, Okla.).

5. Results

[0190] The time course of arterial dimension changes before and afterballoon angioplasty in normal arteries receiving saline infusion wasevaluated in 16 arteries from 8 animals (Table 23). Three segments ofeach artery were studied: the proximal segment immediately upstream fromthe balloon dilated segment, the balloon dilated segment and the distalsegment immediately downstream from the balloon dilated segment. Theproximal and distal segments demonstrated similar patterns of change inarterial dimensions: in each, there was significant change in arterialdiameter when all time points were compared (proximal segment, p=0.0002and distal segment, p<0.001, ANOVA). Post hoc testing indicated that thediameters at the immediate post angioplasty time point weresignificantly less than the diameters at baseline or at the 30 minutetime point in each of these segments. On the other hand, the arterialdiameters in each segment at the 5 minute, 15 minute and 30 minute timepoints were similar to the baseline diameters. The balloon dilatedsegment showed lesser changes in arterial dimension than the proximaland distal segments. The baseline diameter of this segment was 1.82±0.05mm; the nominal inflated diameter of the balloon used for angioplastywas 2.5 mm and the actual measured inflated diameter of the balloon was2.20±0.03 mm (p<0.0001 vs. baseline diameter of the balloon treatedsegment). Thus, the inflated balloon caused circumferential stretch ofthe balloon dilated segment, but there was only slight increase in lumendiameter from baseline to the 30 minute time point (1.82±0.05 mm to1.94±0.07 mm, p=NS by post hoc testing). TABLE 23 Angiographicallydetermined lumen diameters at the specified times before and afterballoon dilatation of normal iliac arteries. Immediate Segment Baseline5 Minute Post PTA 15 Minute 30 Minute Proxima¹ 2.18 ± 0.7 2.03 ± 0.71.81 ± 0.08* 2.00 ± .08 2.23 ± .08 Balloon² 1.82 ± .05 1.77 ± .03 1.79 ±.05  1.70 ± .04 1.94 ± .07 Distal³ 1.76 ± .04  1.68 ± .04** 1.43 ± .04* 1.54 ± .03 1.69 ± .06

[0191] Arterial lumen diameters were used to calculate lumen area thenthe area measurements were used to calculate percent vasoconstriction bycomparison of the 5 minute, immediate post angioplasty, 15 and 30 minutedata to the baseline measurements. The proximal and distal segment dataexpressed as percent vasoconstriction are shown in FIG. 9; the changesin the amount of vasoconstriction over time are significant (in theproximal segment, p=0.0008; in the distal segment, p=0.0001, ANOVA).Post hoc testing identifies the vasoconstriction at the immediate postangioplasty time point as significantly different from that present atthe 30 minute time point (P<0.001 in both segments). In the distalsegment, the immediate post angioplasty vasoconstriction was alsosignificantly less than that at 5 minutes (p<0.01); no other differencesin intra-time point comparisons were significant by post hoc testing.

[0192] The luminal changes in control arteries can be summarized asfollows: 1) Vasoconstriction with loss of approximately 30% of baselineluminal area occurs in the segments of artery proximal and distal to theballoon dilated segment immediately after balloon dilatation. There aretrends to smaller amounts of vasoconstriction in the proximal and distalsegments before dilatation and at the 15 minute time point(approximately 7 minutes after dilatation) also but, by the 30 minutetime point (approximately 22 minutes after dilatation), a trend towardsvasodilatation has replaced the previous vasoconstriction; 2) In theballoon dilated segment, only minor changes in lumen dimensions arepresent, and, despite the use of a balloon with a significantly largerinflated diameter than was present in this segment at baseline, therewas no significant increase in lumen diameter of the dilated segment.These findings lead to a conclusion that any effects of the putativehistamine/serotonin treatment would only be detectable in the proximaland distal segments at the time points where vasoconstriction waspresent.

[0193] The histamine/serotonin receptor blockade solution was infusedinto 16 arteries (8 animals); angiographic data was available at alltime points in 12 arteries. Heart rate and systolic blood pressuremeasurements were available in a subset of animals (Table 24). Therewere no differences in heart rate or systolic blood pressure when thetwo animal groups were compared within specific time points.Histamine/serotonin treated animals showed trends toward a decrease inthe systolic blood pressure from baseline to 30 minutes (−14±5 mm Hg,p=0.04) and a lower heart rate (−26±10, p=0.05). Within the controlanimals, there was no change in heart rate or systolic blood pressureover the duration of the experiment. TABLE 24 Systolic blood pressureand heart rate measurements in control and histamine/serotonin treatedanimals. Baseline 5 Minute 15 Minute 30 Minute Group (N) (N) (N) (N)Systolic Blood Pressure Control 83 ± 4 (8) 84 ± 4 (8) 82 ± 6 (8) 80 ± 4(8) Histamine/Serotonin 93 ± 5 (6) 87 ± 9 (4) 82 ± 9 (6)  80 ± 8 (6)*Heart Rate Control 221 ± 18 (5) 234 ± 18 (4) 217 ± 23 (5) 227 ± 22 (5)Histamine/Serotonin 232 ± 8 (5) 232 ± 8 (5)  209 ± 14 (5)  206 ± 12(5)**

[0194] The proximal and distal segments of histamine/serotonin treatedarteries were compared to control arteries using the percentvasoconstriction measurement. FIG. 10A shows the effects of thehistamine/serotonin infusion on proximal segment vasoconstrictionrelative to the vasoconstriction present in the control arteries. Whenthe findings in the two treatment groups were compared at the baseline,immediate post angioplasty and 15 minute time points,histamine/serotonin infusion resulted in significantly lessvasoconstriction compared to the control saline infusion (p=0.003. 2-wayANOVA). Comparison of the two treatment groups in the distal segment isillustrated in FIG. 10B. Despite observed differences in mean diametermeasurements in the distal segment, solution treated vessels exhibitedless vasoconstriction than saline treated control vessels at baseline,immediate post- angioplasty and 15 minute time points, this pattern didnot achieve statistical significance (p=0.32, 2-way ANOVA). Lack ofstatistical significance may be attributed to smaller than expectedvasoconstriction values in the control vessels.

L. Example XII Amitriptyline Inhibition of 5-Hydroxytryptamine-InducedKnee Joint Plasma Extravasation—Comparison of Intra-Articular VersusIntravenous Routes of Administration

[0195] The following study was undertaken in order to compare two routesof administration of the 5-HT₂ receptor antagonist, amitriptyline: 1)continuous intra-articular infusion; versus 2) intravenous injection, ina rat knee synovial model of inflammation. The ability of amitriptylineto inhibit 5-HT-induced joint plasma extravasation by comparing both theefficacy and total drug dose of amitriptyline delivered via each routewas determined.

1. Animals

[0196] Approval from the Institutional Animal Care Committee at theUniversity of California, San Francisco was obtained for these studies.Male Sprague-Dawley rats (Bantin and Kingman, Fremont, Calif.) weighing300-450 g were used in these studies. Rats were housed under controlledlighting conditions (lights on 6 A.M. to 6 P.M.), with food and wateravailable ad libitum.

2. Plasma Extravasation

[0197] Rats were anesthetized with sodium pentobarbital (65 mg/kg) andthen given a tail vein injection of Evans Blue dye (50 mg/kg in a volumeof 2.5 ml/kg), which is used as a marker for plasma proteinextravasation. The knee joint capsule was exposed by excising theoverlying skin, and a 30-gauge needle was inserted into the joint andused for the infusion of fluid. The infusion rate (250 μl/min) wascontrolled by a Sage Instruments Syringe pump (Model 341B, OrionResearch Inc., Boston, Mass.). A 25-gauge needle was also inserted intothe joint space and perfusate fluid was extracted at 250 μL/min,controlled by a Sage Instruments Syringe pump (Model 351).

[0198] The rats were randomly assigned to three groups: 1) thosereceiving only intra-articular (IA) 5-HT (1 μM), 2) those receivingamitriptyline intravenously (IV) (doses ranging from 0.01 to 1.0 mg/kg)followed by IA 5-HT (1 mM), and 3) those receiving amitriptylineintra-articularly (IA) (concentrations ranging from 1 to 100 nM)followed by IA 5-HT (1 μM) plus IA amitriptyline. In all groups,baseline plasma extravasation levels were obtained at the beginning ofeach experiment by perfusing 0.9% saline intra-articularly andcollecting three perfusate samples over a 15 min period (one every 5min). The first group was then administered 5-HT IA for a total of 25min. Perfusate samples were collected every 5 min for a total of 25 min.Samples were then analyzed for Evans Blue dye concentration byspectrophotometric measurement of absorbance at 620 nm, which islinearly related to its concentration (Carr and Wilhelm, 1964). The IVamitriptyline group was administered the drug during the tail veininjection of the Evans Blue dye. The knee joints were then perfused for15 min with saline (baseline), followed by 25 min perfusion with 5-HT (1μM). Perfusate samples were collected every 5 min for a total of 25 min.Samples were then analyzed using spectrophotometry. In the IAamitriptyline group, amitriptyline was perfused intra-articularly for 10min after the 15 min saline perfusion, then amitriptyline was perfusedin combination with 5-HT for an additional 25 min. Perfusate sampleswere collected every 5 min and analyzed as above.

[0199] Some rat knees were excluded from the study due to physicaldamage of knee joint or inflow and outflow mismatch (detectable bypresence of blood in perfusate and high baseline plasma extravasationlevels or knee joint swelling due to improper needle placement).

a. 5-HT-Induced Plasma Extravasation

[0200] Baseline plasma extravasation was measured in all knee jointstested (total n=22). Baseline plasma extravasation levels were low,averaging 0.022±0.003 absorbance units at 620 nm (average +standarderror of the mean). This baseline extravasation level is shown in FIGS.11 and 12 as a dashed line.

[0201] 5-HT (1 μM) perfused into the rat knee joint produces atime-dependent increase in plasma extravasation above baseline levels.During the 25 min perfusion of 5-HT intra-articularly, maximum levels ofplasma extravasation were achieved by 15 min and continued until theperfusion was terminated at 25 min (data not shown). Therefore,5-HT-induced plasma extravasation levels reported are the average of the15, 20 and 25 min time points during each experiment. 5-HT-inducedplasma extravasation averaged 0.192±0.011, approximately an 8-foldstimulation above baseline. This data is graphed in FIGS. 11 and 12,corresponding to the “0” dose of IV amitriptyline and the “0”concentration of IA amitriptyline, respectively.

b. Effect of Intravenous Amitriptyline on 5-HT-Induced PlasmaExtravasation

[0202] Amitriptyline administered via tail vein injection produced adose-dependent decrease in 5-HT-induced plasma extravasation as shown inFIG. 11. The IC₅₀ for IV amitriptyline inhibition of 5-HT-induced plasmaextravasation is approximately 0.025 mg/kg. 5-HT-induced plasmaextravasation is completely inhibited by an IV amitriptyline dose of Img/kg, the plasma extravasation averaging 0.034±0.010.

c. Effect of Intra-articular amitriptyline on 5-HT-Induced PlasmaExtravasation

[0203] Amitriptyline administered alone in increasing concentrationsintra-articularly did not affect plasma extravasation levels relative tobaseline, with the plasma extravasation averaging 0.018±0.002 (data notshown). Amitriptyline co-perfused in increasing concentrations with 5-HTproduced a concentration-dependent decrease in 5-HT-induced plasmaextravasation as shown in FIG. 12. 5-HT-induced plasma extravasation inthe presence of 3 nM IA amitriptyline was not significantly differentfrom that produced by 5-HT alone, however, 30 nM amitriptylineco-perfused with 5-HT produced a greater than 50% inhibition, while 100nM amitriptyline produced complete inhibition of 5-HT-induced plasmaextravasation. The IC₅₀ for IA amitriptyline inhibition of 5-HT-inducedplasma extravasation is approximately 20 nM.

[0204] The major finding of the present study is that 5-HT (1 μM)perfused intra-articularly in the rat knee joint produces a stimulationof plasma extravasation that is approximately 8-fold above baselinelevels and that either intravenous or intra-articular administration ofthe 5-HT₂ receptor antagonist, amitriptyline, can inhibit 5-HT-inducedplasma extravation. The total dosage of administered amitriptyline,however, differs dramatically between the two methods of drug delivery.The IC₅₀ for IV amitriptyline inhibition of 5-HT-induced plasmaextravasation is 0.025 mg/kg, or 7.5×10⁻³ mg in a 300 g adult rat. TheIC₅₀ for IA amitriptyline inhibition of 5-HT-induced plasmaextravasation is approximately 20 nM. Since 1 ml of this solution wasdelivered every five minutes for a total of 35 min during theexperiment, the total dosage perfused into the knee was 7 ml, for atotal dosage of 4.4×10⁻⁵ mg perfused into the knee. This IAamitriptyline dose is approximately 200-fold less than the IVamitriptyline dose. Furthermore, it is likely that only a small fractionof the IA perfused drug is systemically absorbed, resulting in an evengreater difference in the total delivered dose of drug.

[0205] Since 5-HT may play an important role in surgical pain andinflammation, as discussed earlier, 5-HT antagonists such asamitriptyline may be beneficial if used during the perioperative period.A recent study attempted to determine the effects of oral amitriptylineon post-operative orthopedic pain (Kerrick et al., 1993). An oral doseas low as 50 mg produced undesirable central nervous systemside-effects, such as a “decreased feeling of well-being”. Their study,in addition, also showed that oral arnitriptyline produced higher painscale scores than placebo (P<0.05) in the post-operative patients.Whether this was due to the overall unpleasantness produced by oralamitriptyline is not known. In contrast, an intra-articular route ofadministration allows an extremely low concentration of drug to bedelivered locally to the site of inflammation, possibly resulting inmaximal benefit with minimal side-effects.

M. Example XIII Effects Of Cardiovascular and General Vascular SolutionOn Rotational Atherectomy-Induced Vasospasm In Rabbit Arteries 1.Solution Tested

[0206] This study utilized an irrigation solution consisting of theagents set forth in Example V. above, with the following exceptions.Nitroprusside replaced SIN-1 as the nitric oxide donor and nicardipinereplaced nisoldipine as the Ca²⁺ channel antagonist.

[0207] The concentration of nitroprusside was selected based on itspreviously-defined pharmacological activity (EC₅₀). The concentrationsof the other agents in this test solution were determined based on thebinding constants of the agents with their cognate receptors.Furthermore, all concentrations were adjusted based on a blood flow rateof 80 cc per minute in the distal aorta of the rabbit and a flow rate of5 cc per minute in the solution delivery catheter. Three components weremixed in one cc or less DMSO, and then these components and theremaining three components were mixed to their final concentrations innormal saline. A control solution consisting of normal saline wasutilized. The test solution or the control solution was infused at arate of 5 cc per minute for 20 minutes. A brief pause in the infusionwas necessary at the times blood pressure measurements were made, soeach animal received about 95 cc of the solution in the 20 minutetreatment period.

2. Animal Protocol

[0208] This protocol was approved by the Seattle Veteran Affairs MedicalCenter Committee on Animal Use, which is accredited by the AmericanAssociation for Accreditation of Laboratory Animal Care. The iliacarteries of 3-4 kg male New Zealand white rabbits fed a 2% cholesterolrabbit chow for 3-4 weeks were studied. The animals were sedated usingintravenous xylazine (5 mg/kg) and ketamine (35 mg/kg) dosed to effectand a cutdown was performed in the ventral midline of the neck toisolate a carotid artery. The artery was ligated distally, anarteriotomy performed and a 5 French sheath was introduced into thedescending aorta and positioned at the level of the renal arteries.Baseline blood pressure and heart rate were recorded. An angiogram ofthe distal aorta and bilateral iliac arteries was recorded on 35 mm cinefilm (frame rate 15 per second) using hand injection of iopamidol 76%(Squibb Diagnostics, Princeton, N.J.) into the descending aorta.

[0209] For each angiogram, a calibration object was placed in theradiographic field of view to allow for correction for magnificationwhen diameter measurements were made. Infusion of either the abovedescribed test solution or a saline control solution was started throughthe side arm of the 5 French sheath (and delivered to the distal aorta)at a rate of 5 cc per minute and continued for 20 minutes. At 5 minutesinto the infusion, a second angiogram was performed using the previouslydescribed technique. Then a 1.25 mm or a 1.50 mm rotational atherectomyburr (Heart Technology/Boston Scientific Inc.) was advanced-to the iliacarteries. The rotational atherectomy burr was advanced three times overa guide wire in each of the iliac arteries at a rotation rate of 150,000to 200,000 RPM. In each iliac, the rotational atherectomy burr wasadvanced from the distal aorta to the mid portion of the iliac arterybetween the first and second deep femoral branches. The rotationalatherectomy burr was rapidly removed and another angiogram was recordedon cine film at a mean of 8 minutes after the infusion was begun.

[0210] The infusion was continued until the 20 minute time point, andanother angiogram (the fourth) was performed. Then the infusion wasstopped. A total of about 95 cc of the control or test solution had beeninfused. At the 30 minute time point (15 minutes after the infusion wasstopped), a final angiogram was recorded as before. Blood pressure andheart rate were recorded at the 15 and 30 minute time points immediatelybefore the angiograms. After the final angiogram, the animal waseuthanized with an overdose of the anesthetic agents administeredintravenously.

3. Angiographic Analysis

[0211] The angiograms were recorded on 35 mm cine film at a frame rateof 15 per second. Angiograms were reviewed in random order withoutknowledge of treatment assignment. For analysis, the angiograms wereprojected from a Vanguard projector at a distance of 5.5 feet. Theentire angiogram for each animal was reviewed to identify the anatomy ofthe iliac arteries and to identify the sites of greatest spasm in theiliac arteries. A map of the iliac anatomy was prepared to assist inconsistently identifying sites for measurement. Measurements were madeon the 15 minute post rotational atherectomy angiogram first, then inrandom order on the remaining angiograms from that animal. Measurementswere made using an electronic hand- held caliper (Brown & Sharpe, Inc.,N. Kingston, R.I.). Iliac artery diameters were measured at threelocations: proximal to the first deep femoral branch of the iliacartery; at the site of most severe spasm (this occurred between thefirst and second deep femoral artery branches in all cases); and at adistal site (near or distal to the origin of the second deep femoralartery branch of the iliac artery). Measurements were made at baseline(before test solution infusion was begun), 5 minutes into the infusion,immediately post rotational atherectomy (a mean of 8 minutes after thetest solution was begun), at 20 minutes just after the infusion wasstopped (this was 15 minutes after the rotational atherectomy was begun)and at 15 minutes after the infusion was stopped (30 minutes after therotational atherectomy was begun). The calibration object was measuredin each angiogram.

[0212] The diameter measurements were then converted to areameasurements by the formula:

Area=(Pi)(Diameter²)/4.

[0213] For calculation of vasoconstriction, baseline values were used torepresent the maximum area of the artery and percent vasoconstrictionwas calculated as:

% Vasoconstriction={(Baseline area−Later time point area)/Baselinearea}×100.

4. Statistical Methods

[0214] All values are expressed as mean il standard error of the mean.The time course of vasomotor response in control arteries was assessedusing one way analysis of variance with correction for repeatedmeasures. Post hoc comparison of data between specific time points wasperformed using the Scheffe test. Test solution treated arteries werecompared to saline treated arteries at specified locations in the iliacarteries and at specified time points using multiple analysis ofvariance (MANOVA). To compensate for the absence of a single a priorihypothesis, a p value <0.01 was considered significant. Statistics wereperformed using Statistica for Windows, version 4.5, (Statsoft, Tulsa,Okla.).

5. Results

[0215] Eight arteries in 4 animals received saline solution and 13arteries in seven animals received test solution. In each artery,regardless of the solution used, rotational atherectomy was performedwith the rotating burr passing from the distal aorta to the mid-portionof the iliac artery. Thus, the proximal iliac artery segment and thesegment designated as the site of maximal vasoconstriction weresubjected to the rotating burr. The guide wire for the rotationalatherectomy catheter passed through the distal segment, but the rotatingburr of the rotational atherectomy catheter itself did not enter thedistal segment.

[0216] Iliac artery diameters in saline treated arteries at the threespecified segments are summarized in Table 25. In the proximal segment,there was no significant change in the diameter of the artery over thetime course of the experiment (p=0.88, ANOVA). In the mid-iliac arteryat the site of maximal vasoconstriction, there was a significantreduction in diameter with the largest reduction occurring at the 15minute post-rotational atherectomy time point (p<0.000 1, ANOVAcomparing measurements at all 5 time points). The distal segmentdiameter did not significantly change over the time course of theexperiment (p=0.19, ANOVA comparing all time points) although there wasa trend towards a smaller diameter at the immediate post- and 15 minutepost- rotational atherectomy time points. TABLE 25 Iliac artery lumendiameters at specified time points in saline treated arteries. 5 MinutesImmediate 15 Minute 30 Minutes Baseline into Infusion Post RA after RAafter RA Segment N = 8 N = 8 N = 8 N = 8 N = 8 Proximal¹ 2.40 ± .18 2.32± .14 2.32 ± 0.13 2.38 ± .13  2.34 ± .07* Mid² 2.01 ± .08 1.84 ± .091.57 ± .15  1.24 ± .13  1.87 ± .06** Distal³ 2.01 ± .10 1.86 ± .08 1.79± .08  1.81 ± .09   1.96 ± .06***

[0217] The diameters of iliac arteries treated with the test solutionare shown in Table 26. Angiograms were not recorded in three of thesearteries at the 5 minute post-initiation of the infusion time point andangiographic data were excluded from two arteries (one animal) at the 30minute post-rotational atherectomy time point because the animalreceived an air embolus at the 15 minute angiogram that resulted inhemodynamic instability. Because there is a variable number ofobservations at the five time points, no ANOVA statistic was applied tothis data. Still it is apparent that the magnitude of change in thediameter measurements within segments in the test solution treatedarteries over the time course of the experiment is less than was seen inthe saline treated arteries. TABLE 26 Iliac artery lumen diameters atspecified time points in Test Solution treated arteries. 30 5 MinutesImmediate 15 Minute Minutes Baseline into Infusion Post RA after RAafter RA Segment N = 13 N = 10 N = 13 N = 13 N = 11 Proximal¹ 2.28 ± .062.07 ± .07 2.22 ± .05 2.42 ± .06 2.39 ± .08 Mid² 1.97 ± .06 1.79 ± .061.74 ± .04 1.95 ± .07 1.93 ± .08 Distal³ 2.00 ± .06 1.92 ± .04 1.90 ±.04 2.00 ± .06 2.01 ± .07

[0218] Because of the different number of observations at the varioustime points, ANOVA was not performed to determine the statisticalsimilarity/difference in diameters within specific segments.

[0219] The primary endpoint for this study was the comparison of theamounts of vasoconstriction in saline treated and test solution treatedarteries. Vasoconstriction was based on arterial lumen areas derivedfrom artery diameter measurements. Area values at the 5 minute,immediate post-rotational atherectomy and later time points werecompared to the baseline area values to calculate the relative change inarea. The results were termed “vasoconstriction” if the lumen area wassmaller at the later time point than at baseline, and “vasodilatation”if the lumen area was larger at the later time point compared to thebaseline area (Tables 27 and 28). To facilitate statistical analysiswith the largest number of observations possible in both treatmentgroups, the test solution and saline treated artery data were comparedat the immediate post- and at the 15 minute postrotational atherectomytime points.

[0220] In the proximal segment (FIG. 13), there was essentially nochange in lumen area with either treatment at the immediatepost-rotational atherectomy time point, but there was somevasodilatation in this segment by the 15 minute post-rotationalatherectomy time point. Test solution did not alter the results ofrotational atherectomy compared to saline treatment in this segment. Inthe mid-vessel (FIG. 14) at the site of maximal vasoconstrictionhowever, test solution significantly blunted the vasoconstriction,caused by rotational atherectomy in the saline treated arteries(p=0.0004, MANOVA corrected for repeated measures). In the distalsegment (FIG. 15), there was little vasoconstriction in the salinetreated arteries and test solution did not significantly alter theresponse to rotational atherectomy. TABLE 27 Amount of vasoconstriction(negative values) or vasodilatation (positive values) at specified timepoints in saline treated arteries. 5 Minutes Immediate 15 Minute 30Minutes into Infusion Post RA after RA after RA Segment N = 8 N = 8 N =8 N = 8 Proximal¹ −3% ± .8% −1% ± 10% 3% ± 8% 3% ± 13% Mid² −14% ± 7%−35% ± 10% −58% ± 7% −11% ± .9% Distal³ −9% ± .10% −14% ± .14% −14% ±10% 2% ± .12%

[0221] TABLE 28 Amount of vasoconstriction (negative values) orvasodilatation (positive values) at specified time points in TestSolution treated arteries. 5 Minutes Immediate 15 Minute 30 Minutes intoInfusion Post RA after RA after RA Segment N = 10 N = 13 N = 13 N = 11Proximal¹ −17% ± .5% −4% ± 3% 14% ± 6% 7% ± 9% Mid² −4% ± 5% −20% ± 5%0.3% ± 7% −5% ± .5% Distal³ −8% ± .4% −9% ± .4% 1% ± 4% 3% ± .6%

[0222] The hemodynamic response in the saline and test solution treatedarteries is summarized in Table 29. Compared to saline treated animals,test solution treated animals sustained substantial hypotension andsignificant tachycardia during the solution infusion. By 15 minutesafter completion of the infusion (or at the 30 minute postrotationalatherectomy time point), test solution treated animals showed somepartial, but not complete, return of blood pressure towards baseline.TABLE 29 Blood pressure and heart rates during the protocol. Baseline 5Minute 15 Minute 30 Minute Group (N) (N) (N) (N) Systolic Blood PressureSaline  83 ± 9 (4)  93 ± 6 (3)  92 ± 11 (4)  83 ± 10 (4)* Test  92 ± 5(7)  35 ± 5 (7)  35 ± 5 (7)  46 ± 5 (7)** Solution Heart Rate Saline 202± 16 (3) 204 ± 3 (3) 198 ± 22 (3) 193 ± 29 (3)* Test 187 ± 111 (7) 246 ±11 (7) 240 ± 5 (7) 247 ± 16 (7)** Solution

6. Summary of Study

[0223] 1. Rotational atherectomy in hypercholesterolemic New Zealandwhite rabbits results in prominent vasospasm in the mid-portion of iliacarteries subjected to the rotating burr. The vasospasm is most apparent15 minutes after rotational atherectomy treatment and has almostcompletely resolved without pharmacologic intervention by 30 minutesafter rotational atherectomy.

[0224] 2. Under the conditions of rotational atherectomy treatmentstudied in this protocol, test solution treatment in accordance with thepresent invention almost completely abolishes the vasospasm seen afterthe mid-iliac artery is subjected to the rotating burr.

[0225] 3. Treatment with test solution of the present invention giventhe concentration of components used in this protocol results inprofound hypotension during the infusion of the solution. Theattenuation of vasospasm after rotational atherectomy by test solutionoccurred in the presence of severe hypotension.

[0226] While the preferred embodiment of the invention has beenillustrated and described, it will be appreciated that various changesto the disclosed solutions and methods can be made therein withoutdeparting from the spirit and scope of the invention. For example,alternate pain inhibitors and anti-inflammation and anti- spasm andanti-restenosis agents may be discovered that may augment or replace thedisclosed agents in accordance with the disclosure contained herein. Itis therefor intended that the scope of letters patent granted hereon belimited only by the definitions of the appended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of preemptivelyinhibiting pain and inflammation at a wound during a surgical procedure,comprising: delivering to a wound during a surgical procedure a solutionincluding a plurality of pain/inflamnuation inhibitory agents in aliquid carrier, the plurality of agents being selected to act on aplurality of differing molecular targets, wherein the solution isapplied locally and perioperatively to the surgical site.
 2. The methodof claim 1 , comprising continuously applying the solution to the wound.3. The method of claim 2 , comprising continuously irrigating the woundwith the solution.
 4. The method of claim 1 , wherein the solution isapplied by irrigation of the wound.
 5. The method of claim 1 , whereinthe solution is locally applied to the wound in the absence of metabolictransformation.
 6. The method of claim 1 , wherein the perioperativeapplication of the solution comprises intraprocedural applicationtogether with preprocedural or postprocedural application of thesolution.
 7. The method of claim 6 , wherein the perioperativeapplication of the solution comprises preprocedural, intraprocedural andpostprocedural application of the solution.
 8. The method of claim 6 ,wherein the solution is continuously applied to the wound.
 9. The methodof claim 1 , wherein each of the plurality of agents in the solution isdelivered locally at a concentration of no greater than 100,000nanomolar.
 10. The method of claim 9 , wherein each of the plurality ofagents in the solution is delivered locally at a concentration of nogreater than 10,000 nanomolar.
 11. The method of claim 1 , wherein eachof the plurality of agents in the solution applied is included at aconcentration that is sufficient to provide a predetermined level ofpain/inflammation inhibitory effect at the wound when locally applied inthe absence of metabolic transformation, and that is less than aconcentration which would be required to provide the same predeterminedlevel of inhibitory effect at the wound if applied in a manner whichwould entail metabolic transformation of the agents.
 12. The method ofclaim 1 , wherein the pain/inflammation inhibitory agents are selectedfrom the group consisting of: serotonin receptor antagonists; serotoninreceptor agonists; histamine receptor antagonists; bradykinin receptorantagonists; kallikrein inhibitors; tachykinin receptor antagonistsincluding neurokinin, receptor subtype antagonists and neurokinin₂receptor subtype antagonists; calcitonin gene-related peptide receptorantagonists; interleukin receptor antagonists; phospholipase inhibitorsincluding PLA₂ isoform inhibitors and PLC_(γ) isoform inhibitors;cyclooxygenase inhibitors; lipooxygenase inhibitors; prostanoid receptorantagonists including eicosanoid EP-1 receptor subtype antagonists andeicosanoid EP-4 receptor subtype antagonists and thromboxane receptorsubtype antagonists; leukotriene receptor antagonists includingleukotriene B₄ receptor subtype antagonists and leukotriene D₄ receptorsubtype antagonists; opioid receptor agonists including l-opioidreceptor subtype agonists, 6-opioid receptor subtype agonists, andK-opioid receptor subtype agonists; purinoceptor agonists andantagonists including P_(2Y) receptor agonists and P_(2X) receptorantagonists; and ATP-sensitive potassium channel openers.
 13. The methodof claim 12 , wherein each of the plurality of agents in the solution isdelivered locally at a concentration in nanomolar of from 0.1 to 10,000times the dissociation constant of the agent.
 14. The method of claim 13, wherein each of the plurality of agents in the solution is deliveredlocally at a concentration in nanomolar of from 1.0 to 1,000 times thedissociation constant of the agent.
 15. The method of claim 14 , whereineach of the plurality of agents in the solution is delivered locally ata concentration in nanomolar of 100 times the dissociation constant ofthe agent.
 16. The method of claim 12 , wherein the selectedpain/inflammation inhibitory agents are delivered locally at aconcentration of: 0.1 to 10,000 nanomolar for serotonin receptorantagonists; 0.1 to 2,000 nanomolar for serotonin receptor agonists;0.01 to 1,000 nanomolar for histamine receptor antagonists; 0.1 to10,000 nanomolar for bradykinin receptor antagonists; 0.1 to 1,000nanomolar for kallikrein inhibitors; 0.1 to 10,000 nanomolar forneurokinin, receptor subtype antagonists; 1.0 to 10,000 nanomolar forneurokinin₂ receptor subtype antagonists; 1 to 1,000 nanomolar forcalcitonin gene-related peptide receptor antagonists; 1 to 1,000nanomolar for interleukin receptor antagonists; 100 to 100,000 nanomolarfor PLA₂ isoform inhibitors; 100 to 200,000 nanomolar for cyclooxygenaseinhibitors; 100 to 10,000 nanomolar for lipooxygenase inhibitors; 100 to10,000 nanomolar for eicosanoid EP-1 receptor subtype antagonists; 100to 10,000 nanomolar for leukotriene B₄ receptor subtype antagonists; 0.1to 500 nanomolar for μ-opioid receptor subtype agonists; 0.1 to 500nanomolar for δ-opioid receptor subtype agonists; 0.1 to 500 nanomolarfor K-opioid receptor subtype agonists; 100 to 100,000 nanomolar forpurinoceptor antagonists; and 0.1 to 10,000 nanomolar for ATP-sensitivepotassium channel openers.
 17. The method of claim 1 , wherein thesolution applied comprises: a serotonin₂ receptor subtype antagonistincluded at a concentration of 50 to 500 nanomolar; a serotonin₃receptor subtype antagonist included at a concentration of 200 to 2,000nanomolar; a histamine₁ receptor subtype antagonist included at aconcentration of 5 to 200 nanomolar; a serotonin receptor agonistincluded at a concentration of 10 to 200 nanomolar; a cyclooxygenaseinhibitor included at a concentration of 500 to 5,000 nanomolar; aneurokinin₁ receptor subtype antagonist included at a concentration of10 to 500 nanomolar; a neurokinin₂ receptor subtype antagonist includedat a concentration of 10 to 500 nanomolar; a purinoceptor antagonistincluded at a concentration of 10,000 to 100,000 nanomolar; anATP-sensitive potassium channel opener included at a concentration of100 to 1,000 nanomolar; a calcium channel antagonist included at aconcentration of 100 to 5,000 nanomolar; a bradykinin, receptor subtypeantagonist included at a concentration of 10 to 200 nanomolar; abradykinin₂ receptor subtype antagonist included at a concentration of50 to 500 nanomolar; and a μ-opioid receptor subtype agonist included atconcentration of 10 to 200 nanomolar.
 18. A method of preemptivelyinhibiting pain and inflammation at a wound during a surgical procedure,comprising: delivering to a wound during a surgical procedure a solutionincluding a plurality of pain/inflammation inhibitory agents in a liquidcarrier, the plurality of agents being selected to act on a plurality ofdiffering molecular targets, wherein the solution is appliedperioperatively and in the absence of metabolic transformation to thewound.
 19. A solution for use in the preemptive inhibition of pain andinflammation at a wound during a surgical procedure, comprising aplurality of pain and inflammation inhibitory agents in a liquidcarrier, the concentration of each agent within the solution being theconcentration of that agent which is desired to be delivered locally, inthe absence of metabolic transformation, to a wound in order to achievea predetermined level of inhibitory effect at the wound.
 20. Thesolution of claim 19 , wherein each of the plurality of agents in thesolution is included at a concentration of no greater than 100,000nanomolar, adjusted for dilution in the absence of metabolictransformation, at an intended local delivery site.
 21. The solution ofclaim 20 , wherein each of the plurality of agents in the solution isincluded at a concentration of no greater than 10,000 nanomolar,adjusted for dilution, in the absence of metabolic transformation, at anintended local delivery site.
 22. The solution of claim 19 , whereineach of the plurality of agents in the solution is included at aconcentration that is less than a concentration which would be requiredto provide the same predetermined level of inhibitory effect at thewound if the solution was applied in a manner which would entailmetabolic transformation of the agents.
 23. The solution of claim 19 ,wherein the pain/inflammation inhibitory agents are selected from thegroup consisting of: serotonin receptor antagonists; serotonin receptoragonists; histamine receptor antagonists; bradykinin receptorantagonists; kallikrein inhibitors; tachykinin receptor antagonistsincluding neurokinin, receptor subtype antagonists and neurokinin₂receptor subtype antagonists; calcitonin gene-related peptide receptorantagonists; interleukin receptor antagonists; phospholipase inhibitorsincluding PLA₂ isoform inhibitors and PLC_(γ) isofomi inhibitors;cyclooxygenase inhibitors; lipooxygenase inhibitors; prostanoid receptorantagonists including eicosanoid EP-1 receptor subtype antagonists andeicosanoid EP-4 receptor subtype antagonists and thromboxane receptorsubtype antagonists; leukotriene receptor antagonists includingleukotriene B₄ receptor subtype antagonists and leukotriene D₄ receptorsubtype antagonists; opioid receptor agonists including μ-opioidreceptor subtype agonists, δ-opioid receptor subtype agonists, andκ-opioid receptor subtype agonists; purinoceptor agonists andantagonists including P_(2Y) receptor agonists and P_(2X) receptorantagonists; and ATP-sensitive potassium channel openers.
 24. Thesolution of claim 23 , wherein each of the plurality of agents in thesolution is included at a concentration in nanomolar of from 0.1 to10,000 times the dissociation constant of the agent, adjusted fordilution, in the absence of metabolic transformation, at an intendedlocal delivery site.
 25. The solution of claim 24 , wherein each of theplurality of agents in the solution is included at a concentration innanomolar of from 1.0 to 1,000 times the dissociation constant of theagent, adjusted for dilution, in the absence of metabolictransformation, at an intended local delivery site.
 26. The solution ofclaim 25 , wherein each of the plurality of agents in the solution isincluded at a concentration in nanomolar of 100 times the dissociationconstant of the agent, adjusted for dilution, in the absence ofmetabolic transformation, at an intended local delivery site.
 27. Thesolution of claim 23 , wherein the selected pain/inflammation inhibitoryagents are included at a concentration of: 0.1 to 10,000 nanomolar forserotonin receptor antagonists; 0.1 to 2,000 nanomolar for serotoninreceptor agonists; 0.01 to 1,000 nanomolar for histamine receptorantagonists; 0.1 to 10,000 nanomolar for bradykinin receptorantagonists; 0.1 to 1,000 nanomolar for kallikrein inhibitors; 0.1 to10,000 nanomolar for neurokinin, receptor subtype antagonists; 1.0 to10,000 nanomolar for neurokinin₂ receptor subtype antagonists; 1 to1,000 nanomolar for calcitonin gene-related peptide receptorantagonists; 1 to 1,000 nanomolar for interleukin receptor antagonists;100 to 100,000 nanomolar for PLA₂ isoform inhibitors; 100 to 200,000nanomolar for cyclooxygenase inhibitors; 100 to 10,000 nanomolar forlipooxygenase inhibitors; 100 to 10,000 nanomolar for eicosanoid EP-1receptor subtype antagonists; 100 to 10,000 nanomolar for leukotriene B₄receptor subtype antagonists; 0.1 to 500 nanomolar for i-opioid receptorsubtype agonists; 0.1 to 500 nanomolar for 6-opioid receptor subtypeagonists; 0.1 to 500 nanomolar for K-opioid receptor subtype agonists;100 to 100,000 nanomolar for purinoceptor antagonists; and 0.1 to 10,000nanomolar for ATP-sensitive potassium channel openers.
 28. The solutionof claim 23 , wherein the solution comprises: a serotonin₂ receptorsubtype antagonist included at a concentration of 50 to 500 nanomolar; aserotonin₃ receptor subtype antagonist included at a concentration of200 to 2,000 nanomolar; a histamine, receptor subtype antagonistincluded at a concentration of 5 to 200 nanomolar; a serotonin receptoragonist included at a concentration of 10 to 200 nanomolar; acyclooxygenase inhibitor included at a concentration of 500 to 5,000nanomolar; a neurokinin₁ receptor subtype antagonist included at aconcentration of 10 to 500 nanomolar; a neurokinin₂ receptor subtypeantagonist included at a concentration of 10 to 500 nanorolar; apurinoceptor antagonist included at a concentration of 10,000 to 100,000nanomolar; an ATP-sensitive potassium channel opener included at aconcentration of 100 to 1,000 nanomolar; a calcium channel antagonistincluded at a concentration of 100 to 5,000 nanomolar; a bradykinin,receptor subtype antagonist included at a concentration of 10 to 200nanomolar; a bradykinin₂ receptor subtype antagonist included at aconcentration of 50 to 500 nanomolar; and a μ-opioid receptor subtypeagonist included at concentration of 10 to 200 nanomolar.